U.S. patent application number 11/283483 was filed with the patent office on 2006-07-27 for apparatus and system having dry control gene silencing compositions.
Invention is credited to Anastasia Khvorova, Devin Leake, William S. Marshall, Barbara Robertson, Kathryn Robinson.
Application Number | 20060166234 11/283483 |
Document ID | / |
Family ID | 36461367 |
Filed Date | 2006-07-27 |
United States Patent
Application |
20060166234 |
Kind Code |
A1 |
Robertson; Barbara ; et
al. |
July 27, 2006 |
Apparatus and system having dry control gene silencing
compositions
Abstract
An RTF testing plate can include at least a first control well
including a substantially dry first control composition having at
least a first control siRNA. The first control siRNA is capable of
providing a first indication of the gene silencing efficacy.
Additionally, the first control composition can be configured such
that the first control siRNA is capable of being solubilized or
suspended in an aqueous medium in an amount sufficient for
transfecting cells in the first control well. The control siRNA can
be any one of a transfection control siRNA, positive control siRNA,
or negative control siRNA. Optionally, the total amount of control
siRNA in the first control composition can be present in an amount
for transfecting cells in only the first control well.
Inventors: |
Robertson; Barbara;
(Boulder, CO) ; Leake; Devin; (Denver, CO)
; Robinson; Kathryn; (Golden, CO) ; Marshall;
William S.; (Boulder, CO) ; Khvorova; Anastasia;
(Boulder, CO) |
Correspondence
Address: |
Jonathan M. Benns, Ph.D.;WORKMAN NYDEGGER
1000 Eagle Gate Tower
60 East South Temple
Salt Lake City
UT
84111
US
|
Family ID: |
36461367 |
Appl. No.: |
11/283483 |
Filed: |
November 18, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60630320 |
Nov 22, 2004 |
|
|
|
60678165 |
May 4, 2005 |
|
|
|
Current U.S.
Class: |
435/6.11 ;
435/287.2; 435/6.16 |
Current CPC
Class: |
C12N 2330/31 20130101;
C12N 2320/12 20130101; C12N 2310/14 20130101; C12N 15/111
20130101 |
Class at
Publication: |
435/006 ;
435/287.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/34 20060101 C12M001/34 |
Claims
1. A reverse transfection plate for testing the efficacy of gene
silencing, the plate comprising: at least a first control well
including a substantially dry first control composition having at
least a first control siRNA capable of providing a first indication
of gene silencing efficacy, the first control composition being
configured such that the at least first control siRNA is capable of
being solubilized or suspended in an aqueous medium in an amount
sufficient for transfecting cells in the first control well.
2. A plate as in claim 1, wherein control siRNA is at least one of
a transfection control siRNA, positive control siRNA, or negative
control siRNA.
3. A plate as in claim 2, further comprising at least a second
control well including a substantially dry second control
composition having at least a second control siRNA capable of
providing a second indication of gene silencing efficacy that is
different from the first indication, the second control composition
being configured such that the at least second control siRNA is
capable of being solubilized or suspended in an aqueous medium in
an amount sufficient for transfecting cells in the second control
well.
4. A plate as in claim 2, wherein the positive control siRNA
silences expression of a known gene.
5. A plate as in claim 4, wherein the positive control siRNA
silences expression of at least one of a cyclophilin B, lamin A/C,
or glyceraldehyde-3-phosphate dehydrogenase.
6. A plate as in claim 2, wherein the transfection control siRNA
includes a label.
7. A plate as in claim 6, wherein the label is coupled with a 5'
terminal nucleotide or 3' terminal nucleotide on one of a sense
strand or an antisense strand.
8. A plate as in claim 7, wherein the label is a fluorescent label
on the sense strand.
9. A plate as in claim 2, wherein the transfection control siRNA is
toxic to cells.
10. A plate as in claim 2, wherein the negative control siRNA is
non-functional siRNA.
11. A plate as in claim 10, wherein the non-functional siRNA
includes at least one of a 17 base pair duplex containing a 17 base
pair duplex having 2' modifications on a first and second sense
nucleotide, and having 2' modifications at the first and second
antisense nucleotide, or a 19 base pair duplex having 5' deoxy
nucleotides on sense and antisense 5' terminal nucleotides.
12. A plate as in claim 2, wherein the negative control siRNA
inhibits being taken in and processed by RISC.
13. A plate as in claim 3, wherein the first control composition
includes a positive control siRNA and the second control
composition includes a negative control siRNA.
14. A plate as in claim 3, wherein the first control composition
includes a pool of control siRNAs.
15. A plate as in claim 3, further comprising at least a third
control well including a substantially dry third control
composition having at least a third control siRNA capable of
providing a third indication of gene silencing efficacy that is
different from at least one of the first indication or second
indication, the third control composition being configured such
that the at least third control siRNA is capable of being
solubilized or suspended in an aqueous medium in an amount
sufficient for transfecting cells in the third control well.
16. A plate as in claim 1, further comprising at least one well
devoid of siRNA.
17. A plate as in claim 1, wherein a total amount of control siRNA
in the first control composition is present in an amount for
transfecting cells in only the first control well.
18. A plate as in claim 1, further comprising at least one well
including a substantially dry gene silencing composition, the gene
silencing composition having at least a first test siRNA which
silences a first target gene, the gene silencing composition being
configured such that the at least first test siRNA is capable of
being solubilized or suspended in an aqueous medium in an amount
sufficient for transfecting cells in the well, wherein the gene
silencing composition is characterized by at least one of the
following: the at least first test siRNA is not modified; the at
least first test siRNA has a stabilizing modification; the at least
first test siRNA has a modification to limit off-targeting; the at
least first test siRNA has a conjugate; or the at least first test
siRNA has a hairpin structure.
19. A reverse transfection system for testing the efficacy of gene
silencing, the system comprising: a plate comprising at least a
first control well including a substantially dry first control
composition having at least a first control siRNA, wherein the at
least first control siRNA is capable of being solubilized or
suspended in an aqueous medium in an amount sufficient for
transfecting cells in the well; and a polynucleotide carrier
configured to complex with the at least first control siRNA.
20. A system as in claim 19, wherein the control siRNA is
characterized by one of the following: a positive control siRNA
that silences expression of a known gene; a transfection control
siRNA that includes a fluorescent label; a transfection control
siRNA that includes at least one toxic motif; a negative control
siRNA that is non-functional siRNA; or a negative control siRNA
that inhibits being taken in and processed by RISC.
21. A system as in claim 20, further comprising at least a second
control well including a substantially dry second control
composition having at least a second control siRNA, wherein the at
least second control siRNA is capable of being solubilized or
suspended in an aqueous medium in an amount sufficient for
transfecting cells in the well.
22. A system as in claim 21, further comprising at least a third
control well including a substantially dry third control
composition having at least a third control siRNA, wherein the at
least third control siRNA is capable of being solubilized or
suspended in an aqueous medium in an amount sufficient for
transfecting cells in the well.
23. A system as in claim 21, further comprising at least one well
devoid of siRNA.
24. A method of testing the efficacy of gene silencing with control
siRNA, the method comprising: adding an aqueous medium to a first
control well in a well plate, wherein the first control well
includes a first control siRNA; solubilizing or suspending the
first control siRNA in the aqueous medium; adding cells to the
first well under conditions that permit transfection; and
determining the effect of the first control siRNA on the cells.
25. A method as in claim 24, wherein the aqueous medium includes a
polynucleotide carrier, and further comprising: forming a complex
with the first control siRNA and the polynucleotide carrier,
wherein the complex is suspended or solubilized in the solution;
and contacting the complex to a cell within the first control
well.
26. A method as in claim 24, wherein the control siRNA is
characterized by at least one of the following: a positive control
siRNA that silences expression of known genes; a transfection
control siRNA that includes a fluorescent label; a transfection
second control siRNA is toxic to cells; a negative control siRNA
that is non-functional siRNA; or a negative control siRNA
configured to inhibit being taken in and processed by RISC.
27. A method as in claim 26, further comprising: adding the aqueous
medium to a second control well in the well plate, wherein the
second control well includes a second control siRNA; adding cells
to the second control well under conditions that permit
transfection; and determining the effect of the second control
siRNA on the cells.
28. A method as in claim 27, further comprising: adding the aqueous
medium to a third control well in the well plate, wherein the third
control well includes a third control siRNA; adding cells to the
third control well under conditions that permit transfection; and
determining the effect of the third control siRNA on the cells.
29. A method as in claim 26, further comprising: adding an aqueous
medium to a blank well in the well plate, the blank well being
devoid of siRNA, wherein the aqueous medium optionally includes a
polynucleotide carrier; adding cells to the blank well; and
comparing the effect of the first control siRNA on the cells in the
first control well with the cells added to the blank well.
30. A method as in claim 26, further comprising: adding the aqueous
medium including the polynucleotide carrier to a test well in the
well plate, wherein the test well includes a substantially dry gene
silencing composition, the gene silencing composition having at
least a first test siRNA which silences at least a first target
gene, the gene silencing composition being configured such that the
at least first test siRNA is capable of being solubilized or
suspended in the transfection composition in an amount sufficient
for transfecting cells in the well, wherein the gene silencing
composition is characterized by the following: the at least first
test siRNA is not modified; the at least first test siRNA has a
stabilizing modification; the at least first test siRNA has a
modification to limit off-targeting; the at least first test siRNA
has a conjugate; the at least first test siRNA has a label; or the
at least first test siRNA has a hairpin structure.
31. A method as in claim 26, further comprising: comparing the
effect of the first control siRNA on the cells with a known effect
of the first control siRNA.
32. A method as in claim 25, wherein the polynucleotide carrier is
a lipid.
33. A method as in claim 24, further comprising maintaining the
well plate under conditions so that cell growth, cell division,
transfection, and/or gene silencing occurs.
34. A method as in claim 24, wherein the cells are added in an
amount of about 2.times.10.sup.3 to about 3.times.10.sup.4 cells
per about 0.30 cm.sup.2 to about 0.35 cm.sup.2 of cell growth
surface area.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This United States patent application claims benefit of U.S.
Provisional Application Ser. No. 60/630,320, filed Nov. 22, 2004,
and U.S. Provisional Application Ser. No. 60/678,165, filed May 04,
2005, both of which are incorporated herein by reference.
[0002] This United States Patent Application also cross-references
the following United States patent applications filed herewith:
Attorney Docket No. 16542.1.1, entitled APPARATUS AND SYSTEM HAVING
DRY GENE SILENCING COMPOSITIONS, with Barbara Robertson, Ph.D., et
al. as inventors; Attorney Docket No. 16542.1.2, entitled APPARATUS
AND SYSTEM HAVING DRY GENE SILENCING POOLS, with Barbara Robertson,
Ph.D., et al as inventors; and Attorney Docket No. 16542.1.4,
entitled METHOD OF DETERMINING A CELLULAR RESPONSE TO A BIOLOGICAL
AGENT, with Barbara Robertson, Ph.D., et al. as inventors, wherein
each is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. The Field of the Invention
[0004] The present invention relates to an apparatus and system for
use in RNA interference. More particularly, the present invention
relates to an apparatus and system that include a well plate having
control siRNA.
[0005] 2. The Related Technology
[0006] Recently, a natural cellular regulatory pathway was
discovered that uses transcribed microRNA ("miRNA") in order to
control protein production. The miRNA includes a duplex region of
sense and antisense RNA. This regulatory pathway uses miRNA in
order to target complementary mRNA to inhibit production of the
encoded protein. Accordingly, a complex series of proteins are
involved in this RNA interfering pathway to inhibit or stop
production of the proteins encoded by the mRNA. As such, the
process is referred to as RNA interference or RNAi.
[0007] Additionally, it has been found that the RNAi pathway can be
used with synthetic dsRNA (e.g., siRNA) for silencing genes and
inhibiting protein expression. This can allow for siRNA having
specific sequences to be produced to target complementary DNA
and/or mRNA encoding a specific protein. The siRNA can interact
with the natural RNAi pathway to silence a target gene and inhibit
production of the encoded polypeptide. The ability to silence a
specific gene and inhibit production of the encoded protein has
been used for basic research of gene function, gene mapping,
cellular pathway analysis, and other gene-related studies.
[0008] In order to induce gene silencing, the siRNA needs to be
introduced into a cell. While the most common procedures for
introducing nucleic acids into cells has been forward transfection,
reverse transfection ("RTF") has been developed more recently and
used as an alternative to forward transfection procedures. In
certain versions of RTF protocols, a complex of lipid-nucleic acid
(e.g., lipoplex) can be prepared and introduced into the test wells
of a well plate. Cells are introduced into the test wells with the
lipid-nucleic acid complexes, and incubated so that the siRNA can
enter the cells. Examples of some RTF protocols can be found in
U.S. Pat. No. 5,811,274 to Palsson, U.S. Pat. No. 5,804,431 to
Palsson and U.S. Pat. No. 6,544,790 to Sabatini and in U.S.
Published Applications 2002/0006664 to Sabatini and 2003/070642 to
Caldwell et al. As described in these references, RTF procedures
for nucleic acids generally can have fewer steps compared to
traditional forward transfection and may offer benefits in
attempting to isolate the transfected cells to particular regions
of a single surface, such as a glass slide. However, RTF procedures
for siRNA have not been optimized to the point of practical
application, and improvements in gene silencing efficacy are still
needed, especially for situations in which one is experimenting
with multiple different siRNAs, different gene targets or different
cell lines.
[0009] Often, RTF protocols can be performed on well plates in a
manner that is not optimized or produces inaccurate and unreliable
data. The lack of optimization can cause variations between plates,
and can reduce the reliability of the data. Variations in data from
a lack of optimization or an error can produce results that appear
to be related to the gene silencing obtained from the siRNA, and
are difficult to detect without comparing the test results to other
experiments performed with the same siRNA. Additionally, systematic
variations in data that occur can arise from the RTF conditions,
and may be related to the amount of siRNA, amount of siRNA carrier,
cell density, media, temperature, or various other factors that
affect gene silencing.
[0010] Therefore, it would be advantageous to have an improved RTF
protocol for testing the efficacy of gene silencing. Additionally,
it would be beneficial to have an RTF format that uses controls to
test the efficacy of gene silencing.
BRIEF SUMMARY OF THE INVENTION
[0011] Generally, embodiments of the present invention include well
plates, kits, systems, and methods of using the same for testing
the efficacy of gene silencing. Accordingly, the present invention
provides well plates, kits, and systems that implement an improved
RTF testing protocol for delivering control siRNA into cells to
test the efficacy of gene silencing in other cells in the well
plate or other well plates. The control siRNA can provide an
indication of gene silencing efficacy that can be compared to known
functionalities and standard results obtained from using the
control siRNA in optimal or other test conditions.
[0012] In one embodiment, the present invention can include a
reverse transfection plate for testing the efficacy of gene
silencing. The plate can include at least a first control well
including a substantially dry first control composition having at
least a first control siRNA. The first control siRNA can be capable
of providing a first indication of the gene silencing efficacy.
Additionally, the first control composition can be configured such
that the first control siRNA is capable of being solubilized or
suspended in an aqueous medium in an amount sufficient for
transfecting cells in the first control well. The control siRNA can
be any one of a transfection control siRNA, positive control siRNA,
or negative control siRNA. Optionally, the total amount of control
siRNA in the first control composition can be present in an amount
for transfecting cells in only the first control well.
[0013] In one embodiment, the plate can further include at least a
second control well including a substantially dry second control
composition. The second control composition can include at least a
second control siRNA, which can be any of the transfection,
positive, or negative control siRNAs. Preferably, the second
control siRNA is different from the first control siRNA. As such,
the second control siRNA can provide a second indication of gene
silencing efficacy that is different from the first indication. The
second control composition can be configured such that the second
control siRNA is capable of being solubilized or suspended in an
aqueous medium in an amount sufficient for transfecting cells in
the second control well. Optionally, the first control composition
includes a positive control siRNA and the second control
composition includes a negative control siRNA. Alternatively, the
first control composition includes a positive control siRNA and the
second control composition includes a transfection control siRNA.
In yet another alternative, the first control composition can have
a transfection control siRNA and the second control composition can
include a negative control siRNA.
[0014] In one embodiment, the plate can further include at least a
third control well including a substantially dry third control
composition. The third control composition can include at least a
third control siRNA, which can be any of the transfection,
positive, or negative control siRNA. Preferably, the third control
siRNA is different, from the first control siRNA and second control
siRNA. As such, the third control siRNA can provide a third
indication of gene silencing efficacy that is different from the
first indication and second indication. The third control
composition can be configured such that the third control siRNA is
capable of being solubilized or suspended in an aqueous medium in
an amount sufficient for transfecting cells in the third control
well. Preferably, the first, second, and third control siRNAs
include a transfection, positive, and negative control siRNA,
respectively.
[0015] In one embodiment, the transfection control siRNA, positive
control siRNA, or negative control siRNA can conform to the
descriptions provided herein and in the incorporated
references.
[0016] In one embodiment, the present invention provides a kit or
system that includes a well plate having a control well with a
substantially dry control composition comprised of control siRNA.
Additionally, such a kit or system includes a polynucleotide
carrier. The polynucleotide carrier can be a cationic lipid,
polymer, lipopolymer, or the like. Additionally, the kit or system
can include various solubilizing solutions, reagents, cell culture
media, and the like.
[0017] In one embodiment, the present invention includes a method
of testing the efficacy of gene silencing with control siRNA. This
can include testing the conditions used in the RTF protocol, which
may be related to cell density, type of polynucleotide carrier,
carrier concentration, siRNA concentration, RTF protocol, or other
factors that can alter the effectiveness for siRNA to silence
genes. Additionally, such a testing method can include the use of
any well plate consistent with the foregoing characterizations.
Accordingly, an aqueous medium can be added to a first control well
in the well plate, wherein the first control well includes a first
control siRNA. The aqueous medium can solubilize or suspend the
first control siRNA. Optionally, the aqueous medium can include a
polynucleotide carrier such as a cationic lipid, polymer,
lipopolymer, and the like, which can form a complex with the
control siRNA. The complex can be prepared so as to be capable of
being suspended or solubilized in the aqueous medium.
[0018] Cells can be added to the first well under conditions that
permit transfection with the complex. The cells can be added in an
amount of about 1.times.10.sup.3 to about 3.5.times.10.sup.4 or
about 2.times.10.sup.3 to about 3.times.10.sup.4 cells per about
0.3 cm.sup.2 to about 0.35 cm.sup.2 of cell growth surface area.
Subsequently, the control siRNA can contact the cell in a manner
that allows for entry into the cellular cytoplasm. However, any
mode of transfection can be used to cause the control siRNA to
enter the cell. The well plate can then be maintained under
conditions so that cell growth, cell division, transfection, and/or
gene silencing occurs. After a proper duration that allows for
transfection and/or the control siRNA to silence a known gene, the
effect of the first control siRNA on the cells can be determined.
The effect of the first control siRNA in the cells can be compared
with a known effect of the first control siRNA.
[0019] In one embodiment, a second control siRNA can be used to
test the efficacy of gene silencing. As such, the testing protocol
can include adding the aqueous medium to a second control well in
the well plate, wherein the second control well includes a second
control siRNA. Subsequently, the cells can be added to the second
control well under conditions that permit transfection, and the
second control siRNA can then be transfected into the cells by any
mode of transfection. The effect of the second control siRNA on the
cells can be determined. The effect of the second control siRNA can
be compared to the effect of the first control siRNA.
[0020] In one embodiment, a third control siRNA can be used to test
the efficacy of gene silencing. As such, the testing protocol can
include adding the aqueous medium to a third control well in the
well plate, wherein the third control well includes a third control
siRNA. Subsequently, the cells can be added to the third control
well under conditions that permit transfection, and the third
control siRNA can then be transfected into the cells by any mode of
transfection. The effect of the third control siRNA on the cells
can be determined. The effect of the third control siRNA can be
compared to the effect of the first control siRNA and/or the effect
of the second control siRNA.
[0021] In one embodiment, a blank well that is substantially devoid
of siRNA can be used to test the efficacy of gene silencing. As
such, the testing protocol can include adding an aqueous medium to
a blank well in the well plate, the blank well being devoid of
siRNA. Optionally, the aqueous medium can include the
polynucleotide carrier. Optionally, the cells are then added to the
blank well, and the effects of control siRNA on the cells are
compared to the cells added to the blank well. On the other hand,
the cells may not be added to the blank well so that it can be used
for various calibrations.
[0022] These and other embodiments and features of the present
invention will become more fully apparent from the following
description and appended claims, or may be learned by the practice
of the invention as set forth hereinafter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] To further clarify the above and other advantages and
features of the present invention, a more particular description of
the invention can be rendered by reference to specific embodiments
thereof which are illustrated in the appended drawings. It is
appreciated that these drawings depict only typical embodiments of
the invention and are therefore not to be considered limiting of
its scope. The invention can be described and explained with
additional specificity and detail through the use of the
accompanying drawings.
[0024] FIGS. 1A and 1B are schematic diagrams that illustrate an
embodiment of a multi-well plate having control siRNA and test
siRNA.
[0025] FIGS. 2A-2D are schematic diagrams that illustrate
embodiments of arrangements of control siRNA on a multi-well
plate
[0026] FIGS. 3A-3J are graphical representations of embodiments of
the effects of various plate conditions on siRNA RTF gene silencing
and cell viability. FIG. 3A illustrates cell viability and FIG. 3B
illustrates gene silencing of plain plates. FIG. 3C illustrates
cell viability and FIG. 3D illustrates gene silencing of
poly-L-lysine ("PLL") coated plates. FIG. 3E illustrates cell
viability and FIG. 3F illustrates gene silencing of CELLBIND.TM.
plates. FIG. 3G illustrates cell viability and FIG. 6H illustrates
gene silencing of MATRIGEL.TM. plates. FIG. 3I illustrates cell
viability and FIG. 3J illustrates gene silencing of fibronectin
plates. In cell survival measurements, the Y-axis represents
relative levels of survival with 1.0 being 100% viability. In gene
silencing measurements, the Y-axis represents the level of gene
expression compared to controls with 1.0 being 100% expression, and
"ug" is microgram.
[0027] FIG. 4 is a graphical representation of an embodiment of a
comparison of human cyclophilin B gene silencing at different cell
plating densities. HeLa cells at 10K, 20K, and 40K cells per well
were transfected with cyclo 3, cyclo 28, or cyclo 37 siRNA at
varying concentrations (e.g., 4 nM-250 nM) using DharmaFECT.TM. 1
lipid at 0.063 micrograms ("ug") for 10K cells, 0.125 ug for 20K
cells, and 0.250 ug for 40K cells of per 100 microliters ("uL")
volume, respectively. The total volume in each well is 125 uL. The
figure demonstrates the role that cell density plays in siRNA
silencing efficiency in a reverse transfection format. For gene
silencing, the Y-axis represents the level of gene expression
compared to controls with 1.0 being 100% expression.
[0028] FIGS. 5A-5C are graphical representations of an embodiment
of the identification of toxic siRNA. HeLa cells were forward
transfected at 5,000 cells per well with 10 nM siRNA. FIG. 5A
depicts a DBI-siRNA walk identifying toxic siRNA, wherein the black
bars represent DBI silencing, and the gray bars represent cell
survival. FIG. 5B depicts cell survival resulting from the
introduction of one of forty-eight different siRNA directed against
one of twelve different targets. FIG. 5C depicts an examination of
eight siRNA derived from FIG. 5B, and shows that toxicity is
unrelated to target specific silencing. Also, the data demonstrates
that pooling is one means of eliminating siRNA-induced
toxicity.
[0029] FIGS. 6A-6C are graphical representations of an embodiment
of the identification of toxic motifs responsible for siRNA induced
cell toxicity. FIG. 6A depicts thirty-eight randomly selected siRNA
containing AAA/UUU motifs tested for the ability to induce
toxicity. FIG. 6B depicts nineteen randomly selected siRNA carrying
the GCCA/UGGC motif tested for the ability to induce toxicity. FIG.
6C depicts thirty-two randomly selected siRNA that do not carry
either the AAA/UUU or GCCA/UGGC motifs were tested for toxicity. In
the figures, black bars represent sequences that induce toxicity,
and gray bars represent non-toxic sequences.
[0030] FIG. 7A is a schematic representation of embodiments of
assays to study the involvement of the RNAi pathway in siRNA
induced toxicity, which shows control and experimental studies.
[0031] FIGS. 7B-7I are images of embodiments of green protein
fluorescence resulting from the control and experimental conditions
demonstrating that Ago2 silencing prevents siRNA targeting EGFP
from silencing the intended target. FIG. 7B depicts EGFP expression
pattern and FIG. 7C depicts Hoechst 33342 stained cells to show
that treatment of cells with a control siRNA (e.g.,
siRNA-RISC-Free) in both transfection 1 (T1) and transfection 2
(T2) does not affect EGFP expression. FIGS. 7D and 7E show when T1
is a control siRNA and T2 is an siRNA directed against EGFP, it is
possible to silence EGFP expression. FIGS. 7F and 7G show when T1
uses an siRNA directed against the eIF2C2 protein (e.g., Ago2), but
T2 uses an control siRNA, EGFP expression is maintained. FIGS. 7H
and 7I show when T1 uses an siRNA directed against the eIF2C2
protein, but T2 uses an EGFP-siRNA, EGFP expression is not affected
due to the disruption of the RNAi pathway.
[0032] FIG. 7J is a graphical representation of an embodiment of
testing toxic siRNA in cells that have an intact RNAi pathway with
control siRNA and a disrupted RNAi pathway with siRNA silencing
eIF2C2/Ago2.
[0033] FIG. 8 is a graphical representation of an embodiment of the
effects of truncating toxic siRNA on cell viability.
[0034] FIG. 9A is a graphical representation of an embodiment of
the cell viability of OLIGOFECTAMINE.TM., DharmaFECT.TM. 1 ("DF1"),
and TBio in A549 Cells.
[0035] FIG. 9B is a graphical representation of an embodiment of
the gene silencing of the conditions of FIG. 9A.
[0036] FIG. 10A is a graphical representation of an embodiment of
the cell viability of OLIGOFECTAMINE.TM., DharmaFECT.TM. 1 ("DF1"),
and TBio in 3T3L1 Cells.
[0037] FIG. 10B is a graphical representation of an embodiment of
the gene silencing of the conditions of FIG. 10A.
[0038] FIGS. 11A and 11B are graphical representations of an
embodiment of the gene silencing (FIG. 11A) and cell viability
(FIG. 11B) using three different media/buffer rehydration
solutions, Opti-MEM.TM., HyQ-MEM.TM., and HBSS, in a reverse
transfection format using DharmaFECT.TM. 1 ("DF1"). The numbers
"1", "2", "3", "4", "5" and "8" refer to various biological
replicates performed on different days in this study.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0039] Generally, the present invention is related to an apparatus
and system for use in testing the efficacy of gene silencing in
cells. The apparatus includes plates with wells that have dry
control compositions comprised of control siRNA, which can be
solubilized or suspended for use in RTF testing protocols. The
systems, which can be provided as kits, include the plates and
polynucleotide carriers that can be combined with the control siRNA
to form a transfection complex capable of entering a cell in order
to deliver the control siRNA. Additionally, the system or kits can
include various other solutions and reagents for implementing RTF
protocols.
[0040] The well plates, systems, kits, and methods of the present
invention can be configured for use in high content screening
("HCS") and high throughput screening ("HTS") applications with or
without the use of laboratory automation equipment. Also, the well
plates, systems, kits, and methods can also be used with automated
systems, such as robotic systems. However, the well plates,
systems, kits, and methods can also be used in RTF testing
protocols without the aid of automated delivery systems, or
robotics, and thus can provide an efficient alternative to costly
robotic delivery systems for laboratories using manual processing.
Thus, the well plates, at systems, kits, and methods provide
versatility in choice such that high throughput screening can be
done in a cost effective manner, wherein the efficacy of the gene
silencing used in the screening can be tested along with the test
siRNA.
[0041] The following terminology is defined herein to clarify the
terms used in describing embodiments of the present invention and
is not intended to be limiting. As such, the following terminology
is provided to supplement the understanding of such terms by one of
ordinary skill in the relevant art.
[0042] As used herein, the term "2' modification" is meant to refer
to a chemical modification of a nucleotide that occurs at the
second position atom. As such, the 2' modification can include the
conjugation of a chemical modification group to the 2' carbon of
the ribose ring of a nucleotide, or a nucleotide within an
oligonucleotide or polynucleotide. Thus, a 2' modification occurs
at the 2' position atom of a nucleotide. Examples of a 2'
modification can include a 2'-O-aliphatic, 2'-O-alkyl, 2'-O-methyl,
2'-O-ethyl, 2'-O-propyl, 2'-O-isopropyl, 2'-O-butyl, 2'-O-isobutyl,
2'-O-ethyl-O-methyl (i.e., --CH.sub.2CH.sub.2OCH.sub.3),
2'-O-ethyl-OH (i.e., --OCH.sub.2CH.sub.2OH), 2'-orthoester, 2'-ACE
group orthoester, 2'-halogen, and the like.
[0043] As used herein, the term "antisense strand" is meant to
refer to a polynucleotide or region of a polynucleotide that is at
least substantially (e.g., 80% or more) or 100% complementary to a
target nucleic acid of interest. Also, the antisense strand of a
dsRNA is complementary to its sense strand. An antisense strand may
be comprised of a polynucleotide region that is RNA, DNA, or
chimeric RNA/DNA. Additionally, any nucleotide within an antisense
strand can be modified by including substituents coupled thereto,
such as in a 2' modification. The antisense strand can be modified
with a diverse group of small molecules and/or conjugates. For
example, an antisense strand may be complementary, in whole or in
part, to a molecule of messenger RNA ("mRNA" ), an RNA sequence
that is not mRNA (e.g., tRNA, rRNA, and the like), or a sequence of
DNA that is either coding or non-coding. The antisense strand
includes the antisense region of polynucleotides that are formed
from two separate strands, as well as unimolecular siRNAs that are
capable of forming hairpin structures with complementary base
pairs. The terms "antisense strand" and "antisense region" are
intended to be equivalent and are used interchangeably.
[0044] As used herein, the terms "complementary" and
"complementarity" are meant to refer to the ability of
polynucleotides to form base pairs with one another. Base pairs are
typically formed by hydrogen bonds between nucleotide units in
anti-parallel polynucleotide strands. Complementary polynucleotide
strands can base pair in the Watson-Crick manner (e.g., A to T, A
to U, C to G), or in any other manner that allows for the formation
of duplexes. As persons skilled in the art are aware, when using
RNA as opposed to DNA, uracil rather than thymine is the base that
is considered to be complementary to adenosine.
[0045] Perfect complementarity or 100% complementarity refers to
the situation in which each nucleotide unit of one polynucleotide
strand can hydrogen bond with a nucleotide unit of an anti-parallel
polynucleotide strand. Less than perfect complementarity refers to
the situation in which some, but not all, nucleotide units of two
strands can hydrogen bond with each other. For example, for two
20-mers, if only two base pairs on each strand can hydrogen bond
with each other, the polynucleotide strands exhibit 10%
complementarity. In the same example, if 18 base pairs on each
strand can hydrogen bond with each other, the polynucleotide
strands exhibit 90% complementarity. "Substantial complementarity"
refers to polynucleotide strands exhibiting 79% or greater
complementarity, excluding regions of the polynucleotide strands,
such as overhangs, that are selected so as to be non-complementary.
Accordingly, complementarity does not consider overhangs that are
selected so as not to be similar or complementary to the
nucleotides on the anti-parallel strand.
[0046] As used herein, the term "conjugate" is meant to refer to a
molecule, large molecule, or macromolecular structure that is
coupled with either the sense strand or antisense strand of an
siRNA. That is, the moiety coupled to the siRNA is considered the
conjugate. For clarity purposes, the siRNA can include a conjugate
that is coupled thereto by a covalent bond, ionic interaction, and
like couplings. Usually, a conjugate is coupled with an siRNA in
order to impart a functionality other than increasing the
stabilization or targeting specificity. For examples, some
conjugates, such as cholesterol, can be used to enhance the ability
of the siRNA to enter a cell. Other conjugates can be labels that
can be used to detect transfection or the presence of the siRNA in
the cell. Usually, the conjugate is coupled to the siRNA through a
linker group.
[0047] As used herein, the term "control siRNA" is meant to refer
to a type of siRNA that is well characterized, and can be used to
test the efficacy of gene silencing that can be obtained from an
RTF protocol. As such, a control siRNA can be used alone or with
test siRNA and can provide results that can be compared to known
and established results that have been standardized for that
control siRNA. A control siRNA can be characterized as a positive
control, negative control, and/or a transfection control. Control
siRNA can be used to determine the efficacy of gene silencing that
is being studied with a test siRNA. Control siRNA can be
distinguished from test siRNA in that the control siRNA are well
known and produce reproducable results, and are used as a control
in a study that tests the ability of test siRNA to perform a gene
silencing function, whereas test siRNA can have known or novel
sequences and are usually tested for functionality against a target
gene. Control siRNA are described in more detail below.
[0048] As used herein, the terms "dried" or "dry" as used in
connection with gene silencing compositions is meant to refer to a
composition that is not fluidic and does not flow. However, this
does not exclude small amounts of water or other solvents, and
includes amounts of water remaining in an RNA preparation that has
equilibrated at standard or ambient conditions, for example, at one
atmosphere of pressure, room temperature, and ambient humidity,
such that the preparation is not in a substantially liquid form but
instead is "dried" in the well. For example, an siRNA preparation
is "dried" or substantially "dry" if, at about one atmosphere
pressure, at about 20 to 40.degree. C., and at about 50 to about
95% humidity, the preparation is equilibrated and, when the well
plate is inverted or tilted to, for example, 90.degree. from
horizontal, the RNA preparation does not displace or flow within
the well. This is in comparison to a liquid preparation which would
flow or run when tilted. In various embodiments, methods for using
the dry gene silencing composition in order to perform a
transfection can include solubilizing or suspending the dried
preparation in a suitable aqueous medium to form a mixture.
Additionally, the suitable aqueous medium can include a
polynucleotide carrier capable of facilitating introduction of the
siRNA into a cell, and exposing the mixture to one or more cells to
achieve transfection.
[0049] As used herein, the term "duplex region" is meant to refer
to the region in two complementary or substantially complementary
polynucleotides that form base pairs with one another, either by
Watson-Crick base pairing or any other manner that allows for a
stabilized duplex between the polynucleotide strands. For example,
a polynucleotide strand having 21 nucleotide units can base pair
with another polynucleotide of 21 nucleotide units, yet only 19
bases on each strand are complementary such that the "duplex
region" has 19 base pairs. The remaining bases may, for example,
exist as 5' and/or 3' overhangs. Further, within the duplex region,
100% complementarity is not required, and substantial
complementarity is allowable within a duplex region. Substantial
complementarity refers to 79% or greater complementarity and can
result from mismatches and/or bulges. For example, a single
mismatch in a duplex region consisting of 19 base pairs results in
94.7% complementarity, rendering the duplex region substantially
complementary.
[0050] As used herein, the term "functionality" is meant to refer
to the level of gene specific silencing induced by an siRNA. In
general, functionality is expressed in terms of percentages of gene
silencing. Thus, 90% silencing of a gene (e.g., F90) refers to
situations in which only 10% of the normal levels of gene
expression are observed. Similarly, 80% silencing of a gene (e.g.,
F80) refers to situations in which only 20% of the normal levels of
gene expression are observed.
[0051] As used herein, the term "gene silencing" is meant to refer
to a process by which the expression of a specific gene product is
inhibited by being lessened, attenuated, and/or terminated. Gene
silencing can take place by a variety of pathways. In one instance,
gene silencing can refer to a decrease in gene product expression
that results from the RNAi pathway, wherein an siRNA acts in
concert with host proteins (e.g., RISC) to degrade mRNA in a
sequence-dependent manner. Alternatively, gene silencing can refer
to a decrease in gene product expression that results from siRNA
mediated translation inhibition. In still another alternative, gene
silencing can refer to a decrease in gene product expression that
results from siRNA mediated transcription inhibition. The level of
gene silencing can be measured by a variety of methods, which can
include measurement of transcript levels by Northern Blot Analysis,
B-DNA techniques, transcription-sensitive reporter constructs,
expression profiling (e.g., DNA chips), and related technologies
and assays. Alternatively, the level of gene silencing can be
measured by assessing the level of the protein encoded by a
specific gene that is translated from the corresponding mRNA. This
can be accomplished by performing a number of studies including
Western Blot analysis, measuring the levels of expression of a
reporter protein, such as calorimetric or fluorescent properties
(e.g., GFP), enzymatic activity (e.g., alkaline phosphatases), or
other well known analytical procedures.
[0052] As used herein, the term "nucleotide" is meant to refer to a
ribonucleotide, a deoxyribonucleotide, or modified form thereof, as
well as an analog thereof. Nucleotides include species that
comprise purines, e.g., adenine, hypoxanthine, guanine, and their
derivatives and analogs, as well as pyrimidines, e.g., cytosine,
uracil, thymine, and their derivatives and analogs. Nucleotide
analogs include nucleotides having modifications in the chemical
structure of the base, sugar and/or phosphate, including, but not
limited to, 5'-position pyrimidine modifications, 8'-position
purine modifications, modifications at cytosine exocyclic amines,
and 2'-position sugar modifications (e.g., 2' modifications). Such
modifications include sugar-modified ribonucleotides in which the
2'-OH is replaced by a group such as an H, OR, R, halo, SH, SR,
NH.sub.2, NHR, NR.sub.2, or CN, wherein R is an alkyl or aliphatic
moiety. Nucleotides are well known in the art. Also, reference to a
first nucleotide or nucleotide at a first position refers to the
nucleotide at the 5'-most position of a duplex region, and the
second nucleotide is the next nucleotide toward the 3' end. In
instances the duplex region extends to the end of the siRNA, the 5'
terminal nucleotide can be the first by nucleotide.
[0053] As used herein, the term "polynucleotide" is meant to refer
to polymers of nucleotides linked together through internucleotide
linkages. Also, a polynucleotide includes DNA, RNA, DNA/RNA,
hybrids including polynucleotide chains of regularly and/or
irregularly alternating deoxyribosyl moieties and ribosyl moieties
(i.e., wherein alternate nucleotide units have an --OH, then and
--H, then an --OH, then an --H, and so on at the 2' position of a
sugar moiety), and modifications of these kinds of polynucleotides.
Also, polynucleotides include nucleotides with various
modifications or having attachments of various entities or moieties
to the nucleotide units at any position.
[0054] As used herein, the terms "rational design" and "rationally
designed" are meant to refer to the selection or design of one or
more siRNA(s) for use in a gene silencing application based upon
one or more criteria that are independent of the target sequence.
As such, rationally designed siRNA are selected to specifically
interact with and inhibit polypeptide translation from a selected
mRNA. Thus, for any one target mRNA there may be hundreds of
potential siRNA having from 18 to 31 base pairs that are 100%
complementary to the target mRNA. In part, this is because a single
mRNA may have multiple sequences that can be specifically targeted
by the siRNA. However, it is likely that not all of the siRNA will
have equal functionality. Through empirical studies, a number of
other factors including the presence or absence of certain
nitrogenous bases at certain positions, the relative GC content,
and the like, can affect the functionality of particular siRNA.
Additional information regarding rationally designed siRNA can be
found in commonly owned U.S. patent application Ser. No.
10/714,333, filed on Nov. 14, 2003, related PCT application
PCT/US03/36787, published on Jun. 3, 2004 as WO 2004/045543 A2,
U.S. patent application Ser. No. 10/940,892, filed on Sep. 14,
2004, published as U.S. Patent Application Publication
2005/0255487, related PCT application PCT/US 04/14885, filed on May
12, 2004, and U.S. Patent Application Publication 2005/0246794,
which are all incorporated herein by reference.
[0055] As used herein, the term "reverse transfection" and
abbreviation "RTF" are each meant to refer to a process for
introducing nucleic acid, such as an siRNA, into a cell. Such an
introduction of an siRNA into a cell can be accomplished by
combining the nucleic acid and cell in a well, wherein the cell has
not yet been previously adhered or maintained on the growth
surface. The reverse transfection proceeds by contacting the
nucleic acid onto a cellular surface in a manner such that the
nucleic acid can enter into the cell. Usually, the siRNA is
complexed with a lipid or other polynucleotide carrier prior to
being contacted to the cells. Reverse transfection differs from
forward transfection because the cells have not been seeded and
maintained on the cellular growth surface of a well or other
container before addition of the siRNA.
[0056] As used herein, the term "sense strand" is meant to refer to
a polynucleotide or region that has the same nucleotide sequence,
in whole or in part, as a target nucleic acid such as a messenger
RNA or a sequence of DNA. The term "sense strand" includes the
sense region of a polynucleotide that forms a duplex with an
antisense region of another polynucleotide. Also, a sense strand
can be a first polynucleotide sequence that forms a duplex with a
second polynucleotide sequence on the same unimolecular
polynucleotide that includes both the first and second
polynucleotide sequences. As such, a sense strand can include one
portion of a unimolecular siRNA that is capable of forming hairpin
structure, such as an shRNA. When a sequence is provided, by
convention, unless otherwise indicated, it is the sense strand or
region, and the presence of the complementary antisense strand or
region is implicit. The phrases "sense strand" and "sense region"
are intended to be equivalent and are used interchangeably.
[0057] As used herein, the term "siRNA" is meant to refer to a
small inhibitory RNA duplex that induces gene silencing by
operating within the RNA interference ("RNAi") pathway. These siRNA
are dsRNA that can vary in length, and can contain varying degrees
of complementarity between the antisense and sense strands, and
between the antisense strand and the target sequence. Each siRNA
can include between 17 and 31 base pairs, more preferably between
18 and 26 base pairs, and most preferably 19 and 21 base pairs.
Some, but not all, siRNA have unpaired overhanging nucleotides on
the 5' and/or 3' end of the sense strand and/or the antisense
strand. Additionally, the term "siRNA" includes duplexes of two
separate strands, as well as single strands that can form hairpin
structures comprising a duplex region, which may be referred to as
short hairpin RNA ("shRNA").
[0058] As used herein, the terms "siRNA pool," "pool," "pool of
siRNAs," and "pool reagents" are meant to refer to two or more
siRNA, typically four siRNA, directed against a single target gene,
mRNA, and/or translation of a protein. The siRNA of the pool
reagent can be rationally designed by being selected according to
non-target specific criteria as described herein and in the
incorporated references. For example, two nanomoles of each pool
reagent can be sufficient for transfecting cells in about 200 wells
of multiple 96-well plates, using 100 nM siRNA concentration. Pool
reagents can be plated as a pool (i.e., the two or more siRNA of
Dharmacon's SMARTpool.RTM. Reagent in a single transfection well).
The individual siRNAs that comprise the SMARTpool.RTM. Reagent can
also be plated individually on the same plate as the SMARTpool.RTM.
Reagent.
[0059] As used herein, the term "target" is used in a variety of
different forms throughout this document and is defined by the
context in which it is used. The term "target gene" is meant to
refer to the gene that encodes the protein to be silenced by the
siRNA, and encodes for the production of the target mRNA. The term
"target mRNA" is meant to refer to an mRNA against which a given
siRNA is direct to silence the transcription of the polypeptide
product. The term "target sequence" and "target site" are meant to
refer to a sequence within the mRNA, miRNA, or DNA coding or
promoter region to which the sense strand of an siRNA exhibits
varying degrees of homology and the antisense strand exhibits
varying degrees of complementarity. The term "target polypeptide"
or "target protein" is meant to refer to the gene product encoded
by the target gene, target mRNA, and/or target sequence. The term
"siRNA target" can refer to the gene, mRNA, or protein against
which the siRNA is directed to for silencing. Similarly, "target
silencing" can refer to the state of silencing a gene, or the
corresponding mRNA or protein.
[0060] As used herein, the term "transfection" is meant to refer to
a process by which nucleic acids are introduced into a cell. The
list of nucleic acids that can be transfected is large and
includes, but is not limited to, siRNA, shRNA, sense and/or
anti-sense sequences, DNA, RNA, and the like. There are multiple
modes for transfecting nucleic acids into a cell including, but not
limited to, electroporation, calcium phosphate delivery,
DEAE-dextran delivery, lipid delivery, polymer delivery, molecular
conjugate delivery (e.g., polylysine-DNA or -RNA conjugates,
antibody-polypeptide conjugates, antibody-polymer conjugates,
cholesterol conjugates, or peptide conjugates), microinjection,
laser- or light-assisted microinjection, optoporation or
photoporation with visible and/or nonvisible wavelengths of
electromagnetic radiation, and the like. Transfections can be
"forward transfections" whereby cells are first plated in wells and
then treated with a nucleic acid or they can be "reverse
transfections" (RTF) whereby the nucleic acid is combined with the
cells before or during being plated and/or attached to the bottom
of the well. Any mode of transfecting cells, such as those
described above, can be used with the present invention by inducing
the nucleic acid to be introduced into a cell after the siRNA is
solubilized or suspended in the aqueous medium to implement reverse
transfection. Details regarding a mode of reverse transfection are
described in more detail below
[0061] As used herein, the term "well plate" is meant to refer to a
substrate that is divided into distinct regions that prevent
migration from one distinct region to another distinct region,
wherein the distinct regions are wells. For example, each well of a
multi-well well plate may contain a horizontal well floor that may
be curved or flat, as well as have sidewalls. Additionally, well
plates are well known in the art.
[0062] The use of units to define measurable quantities of
material, such as concentration, weight, and volume, are intended
to be those that are routinely employed by those of skill in the
art. Additionally, the units are preferably interpreted to
correspond with the metric system. Also, the use of "u," as in "ug"
or "uL" is meant to refer to "micro" as applied to microgram and
microliter, respectively.
[0063] Additionally, while the foregoing term definitions are
intended to supplement the knowledge of one of ordinary skill in
the art, not every term within this document has been defined. As
such, the undefined terms are intended to be construed with the
knowledge of one of ordinary skill in the art and/or the plain
meaning of the term. Additionally, the foregoing terms are not
intended to be limited by the examples provided therein, but are
intended to be useful in understanding and practicing the invention
as described herein.
I. Reverse Transfection
[0064] Generally, the present invention provides well plates,
systems, kits, and methods for testing and/or optimizing the
efficacy of procedures and/or conditions that implement reverse
transfection ("RTF") of siRNA. As such, the present invention can
provide for plates, systems, kits, and methods that can be used to
assess or test the efficacy of gene silencing. The present
invention provides methods of determining or assessing the efficacy
of gene silencing so that improvements in both reverse transfection
methodologies that pertain to siRNA and the efficiency of siRNA
based gene silencing can be obtained. In part, this is because the
plates, systems, kits, and methods use control siRNA that can
provide meaningful results pertaining to the accuracy of results
obtained from a gene silencing protocol. Thus, the results obtained
from using control siRNA can be used to validate or invalidate gene
silencing results, and lead to the improvement of siRNA RTF
protocols and the conditions used therewith.
[0065] In one embodiment, the present invention includes a method
of testing and/or optimizing the effectiveness of an siRNA RTF
protocol and/or condition for introducing siRNA into a cell to
effect gene silencing. Such a method can include providing a well
plate that includes a well having a substantially dry control
composition. The control composition can include an individual
control siRNA or a a pool of control siRNAs. The control
composition can include a control siRNA which can be used to
provide meaningful information regarding the effectiveness of the
siRNA RTF protocol and/or condition. Such meaningful information
can provide an indication of whether or not the RTF protocol and/or
condition is sufficient for introducing test siRNA into cells to
effect gene silencing. Also, the meaningful information can provide
an indication of whether or not any gene silencing data obtained
from the siRNA RTF protocol is valid and reliable, or whether the
data inaccurately represents the effectiveness of a test siRNA to
silence a target gene. Also, the control siRNA can provide an
indication of whether any gene silencing is a function of cellular
toxicity or non-specific gene silencing rather than that resulting
from specific gene silencing. Moreover, the method of testing the
effectiveness of an siRNA RTF protocol and/or condition can be used
as part of an optimization procedure so that the optimum gene
silencing conditions can be selected for certain siRNA, cells,
polynucleotide carriers, and the like.
[0066] The control siRNA can be present in the well of a plate as
part of a dry control composition so that the test plates or
optimization plates can be prepared, sealed, stored, and/or shipped
long before an RTF testing protocol is performed. In part, this is
because the dry control composition can stably retain the control
siRNA in a usable condition within the well, and be resuspended or
resolubilized with an aqueous medium during the RTF testing
protocol. Thus, a well plate having the control composition can be
manufactured and hermetically sealed in an inert environment within
a sterile package, wherein the plate can include different wells
with predefined types of control siRNA and optionally test siRNA
for specific gene targets. Such types of control siRNA intended to
test the effectiveness of an siRNA RTF protocol are described in
more detail below.
[0067] In any event, the testing can be performed by adding an
aqueous medium to each well that contains a control composition so
as to suspend or solubilize the control siRNA into the aqueous
medium. The aqueous medium is allowed to solubilize or suspend the
control siRNA for a sufficient duration so that most, if not all,
of the control siRNA is solubilized or suspended. Optionally, the
aqueous medium or an additional solution is comprised of a
polynucleotide carrier. However, polynucleotide carriers are not
necessary in some embodiments.
[0068] After the control siRNA is adequately solubilized or
suspended, cells are added to the well under conditions that permit
the control siRNA to be introduced into the cell. The cells can be
added in an amount of about 1.times.10.sup.3 to about
3.5.times.10.sup.4 cells per about 0.3 cm.sup.2 to about 0.35
cm.sup.2 of cell growth surface area. The conditions that promote
the control siRNA entering the cell can be described by typical
cell culture techniques used for plating cells that are well known
in the art, and further can be the conditions used in an siRNA RTF
protocol to be tested. That is, the cells can be added to the well
that contains the control siRNA in a manner similar to ordinary
plating. Optionally, the cells are added to a well having a dry
control composition so that the aqueous medium carrying the cells
can solubilize or suspend the control siRNA. The well containing
the control siRNA and cells can be incubated for a sufficient
duration for gene silencing to occur, which is typically less than
72 hours, more preferably less than 48 hours, and most preferably
about 24 hours or less.
[0069] In one embodiment, the methods include transfecting the
cells with the siRNA. As such, any mode of transfection can be
implemented in the RTF format by adding the cells to the well
having the siRNA. Accordingly, the cells can be transfected while
suspended or while attaching to the well floor. The modes of
transfection can include those described above or others known or
developed later.
[0070] In one embodiment, the RTF testing protocol can include
adding the polynucleotide carrier to the well so as to form a
control complex, wherein the control complex is suspended or
solubilized in the aqueous medium. After the cells are added, the
control complex can be contacted to the cell to induce endocytosis
of the complex. As such, the polynucleotide carrier can be added as
part of the aqueous medium or in addition thereto. Thus, the
polynucleotide carrier can be presented in an aqueous medium and be
either solubilized or suspended therein. The polynucleotide carrier
can be a cationic lipid, polymer, lipopolymer, and the like.
[0071] After the cells are combined with the control siRNA, the
well plate can be maintained under conditions so that cell growth,
cell division, transfection, and/or gene silencing occurs. Usually,
the cells are maintained in the presence of the control siRNA for
about 6 to about 72 hours before gene silencing is assessed, more
preferably about 12 to about 36 hours, and most preferably for
about 24 to about 48 hours. However, it should be recognized that
the cells are incubated with the control siRNA for a time period
sufficient for silencing a gene so that the amount corresponding
gene product decreases. As such, the production of a target
polypeptide can be silenced by at least about 50%, more preferably
by at least about 70%, even more preferably by at least about 80%,
and most preferably by at least about 90%.
[0072] In instances where cells that grow in suspension are the
target cell, such cells can be added to the wells at an appropriate
cell density and plates can be spun under low gravity forces that
are not detrimental to cell viability to bring the cells and lipids
into close proximity on the bottom of the well.
[0073] In one embodiment, the control composition includes a
positive control siRNA. The positive control siRNA can be
characterized by being capable of silencing expression of a known
gene. Also, the positive control siRNA can provide consistent,
reproducible, and known results that can be used as assess the gene
silencing efficacy of an RTF protocol and/or condition. For
example, the positive control siRNA can silence at least one of a
MAP kinase gene, cyclophilin B gene, lamin A/C gene,
glyceraldehyde-3-phosphate dehydrogenase gene or other well-known
and established control genes that can be reproducibly silenced
[0074] In one embodiment, the control composition includes a
transfection control siRNA. The transfection control siRNA can
provide the ability to assess the level or percentage of cells that
have been successfully transfected with the transfection control
siRNA. That is, a transfection control siRNA can be configured to
identify whether or not it has successfully entered into a cell. In
one aspect, the transfection control siRNA includes a label that
can be detected in a cell. This allows the cells to be analyzed to
determine whether or not the transfection control siRNA is present
in the cell. Examples of labels include colorimetric labels,
chemiluminescent labels, fluorescent labels, mass labels, and
radioactive labels. The labels can be considered conjugates and can
be coupled with siRNA as described for conjugates herein and in the
incorporated references. This includes a direct coupling to the
siRNA and coupling through a linker. The label can be coupled to a
5' terminal nucleotide or 3' terminal nucleotide on one of a sense
strand or an antisense strand. Optionally, the label can be
attached to any nucleotide on the sense strand or antisense strand.
However, it is preferable for the label to be fluorescent, and be
coupled to the sense strand.
[0075] In one embodiment, the transfection control siRNA can be
toxic to cells. This can include siRNA that include at least one
toxic motif. That is, the siRNA includes a polynucleotide sequence
that can lead to cellular toxicity and/or cell death. Examples of
such toxic motifs can include siRNA that include a polynucleotide
sequence including an AAA motif, UUU motif, GCCA motif, or UGGC
motif. However, other toxic siRNA can be employed as transfection
control siRNA.
[0076] In one embodiment, the control composition includes negative
control siRNA. The negative control siRNA can be configured to be
non-functional siRNA. That is, the siRNA can include a
polynucleotide sequence that does not function in the RNAi pathway.
For example, the negative control siRNA can have a polynucleotide
sequence that does not target any known human gene, animal gene, or
gene within the cell being transfected and studied. Also, a
non-functional siRNA can include a 17 base pair duplex containing a
sense strand with 2' modifications at the first and second
nucleotide, and an antisense strand with 2' modifications at the
first and second nucleotide. Alternatively, the non-functional
siRNA can include a 19 base pair duplex having 5' deoxy nucleotides
on the 5' terminal nucleotides of the sense and/or antisense
strands.
[0077] In one embodiment, the negative control siRNA can be
configured to inhibit RISC uptake and processing. This can include
a chemically modified siRNA that inhibits being taken in or
processed by RISC.
[0078] In one embodiment, the cells transfected with the control
siRNA during the RTF testing protocol can be assessed for cell
viability, control gene silencing, control polypeptide production,
control mRNA amount, presence of the control siRNA,
non-functionality of the control siRNA, and the like. The cell
viability studies can be performed in the well plate in accordance
with well known procedures. Additionally, the control gene
silencing can also be assessed with the contents in the well by
various techniques well known in the art to assess the presence or
absence of target proteins. When the control siRNA includes a
label, it can be detected in the cell. Alternatively, the amount of
gene silencing can be assessed by removing the contents from the
well by well known assays. In various embodiments, the well is
designed to be compatible with optical detection systems such as,
for example, UV, luminescence, fluorescence, or light scattering
detection systems, which can be favorable for transfection control
siRNA having fluorescent labels. In embodiments compatible with
optical detection systems, the walls of the well can be made
opaque, or rendered such that light scattering that can interfere
with optical detection is reduced or minimized.
[0079] In one embodiment, the results of the RTF protocol to induce
gene silencing can be detected or monitored using systems for
performing high content screening ("HCS") or high throughput
screening ("HTS.revreaction.). An HCS analysis can be used to
measure specific translocation and morphology changes, receptor
trafficking, cytotoxicity, cell mobility, cell spreading, and the
like. HCS studies can be performed on an ArrayScan.RTM. HCS Reader,
or a KineticScan.RTM. HCS Reader (Cellomics, Inc.) Additional
information on HCS can be found in U.S. Pat. Nos. 6,902,883,
6,875,578, 6,759,206, 6,716,588, 6,671,624, 6,620,591, 6,573,039,
6,416,959, 5,989,835, wherein each is incorporated herein by
reference. HTS analyses can be performed using a variety of
available readers, typically of the fluorescence from each well as
a single measurement.
[0080] In one embodiment, the invention includes a well plate
configured for having the contents of a well transferred to a
location, device, or system wherein detection of the results of an
siRNA RTF testing protocol is carried out. As such, wet transfer
detection systems can be employed that include systems wherein
cells are transferred from wells to a substrate such as
nitrocellulose. Following the transfer of the well contents to the
substrate a detection protocol can be implemented. An example of
such a well plate transfer system can include nitrocellulose,
wherein the well contents can be treated such that cell membranes
are permeabilized or disrupted so as to gain access to
intracellular contents. The transfer of the well contents to the
nitrocellulose can be achieved by any suitable method including
gravity or use of a vacuum manifold. The nitrocellulose containing
the well contents can then be further subjected to a detection
protocol that uses antibody-based detection systems and the like to
detect the presence or level of one or more contents of the cells
that comprise a particular well.
II. Optimizing siRNA RTF
[0081] Due to the unique and highly sensitive nature of the RNAi
pathway, methodologies particularly useful for introducing siRNA
into cells have been developed. Recently developed protocols for
implementing siRNA RTF were modified by augmenting such protocols
with recently developed siRNA technologies based on rationale
design, siRNA stabilization, siRNA targeting specificity, and
pooling siRNAs. However, the effectiveness of new siRNA RTF
protocols can still be compromised by a lack of optimization,
errors, or improper conditions. Thus, methods for testing and
optimizing the efficacy of gene silencing with siRNA RTF formats
are presented herein.
[0082] It has recently been discovered that there are many diverse
responses when different cell numbers, different lipids, and
different lipid to siRNA ratios are used in an RTF format in
comparison with those that are recommended for forward transfection
protocols. Accordingly, control siRNA can be used in RTF testing
protocols to assess the conditions in which a test siRNA is
employed.
[0083] The number of cells per well, which is referred to as the
cell density, is an important parameter of successful siRNA pool
RTF. It has been found that siRNA pool RTF protocols can have more
favorable results with lower cell densities compared to RTF
protocols using DNA. For example, 96-well plates can include cell
densities of about 1,000-35,000 cells per well, more preferably
about 1,250-30,000 cells per well, even more preferred are cell
densities of about 1,500-20,000 cells per well, still more
preferably about 1,750-15,000 cells per well, and most preferable
are cell densities of about 3,000-10,000 cells per well. Also, the
number of cells per well can be extrapolated to wells having
different cell culture areas. One possible equation for calculating
the appropriate number of cells that are placed in a given well is
based on a 96-well plate having a cell culture area of about 0.3
cm.sup.2 to about 0.35 cm.sup.2, wherein well #2 is the 96-well
plate, and is described as follows: cells .times. .times. in
.times. .times. well .times. .times. #1 = ( area .times. .times. of
.times. .times. well .times. .times. #1 area .times. .times. of
.times. .times. well .times. .times. #2 ) .times. cells .times.
.times. in .times. .times. well .times. .times. #2 ##EQU1##
[0084] Additionally, siRNA RTF testing protocols can be used to
determine whether a particular polynucleotide carrier, such as a
lipid, can be useful in a particular siRNA RTF condition. The
polynucleotide carrier can be tested over a wide range of carrier
concentrations by using a robust and easily-transfected cell line
(e.g., HeLa) with control siRNA over commonly used ranges of cell
density and siRNA concentrations. Accordingly, cell viability and
the function of the control siRNA can be assayed with the foregoing
concentration gradients. Thus, optimization studies can be
performed with concentration gradients in order to determine which
polynucleotide carriers can produce highly efficient transfection
without inducing unfavorable cell toxicity.
[0085] In one embodiment, the present invention is directed to
optimization of siRNA RTF protocols for implementing gene silencing
through the RNAi pathway. As such, optimization of siRNA RTF can
include any of the following: (1) selecting the type of plate; (2)
selecting an appropriate solution to solubilize or suspend the
siRNA for being deposited and dried in a well; (3) selecting at
least one control siRNA or a pool of control siRNAs, which can be a
transfection control, positive control, or a negative control; (4)
identifying any modifications or conjugates that can be applied to
the individual siRNA in order to enhance siRNA stability and/or
specificity; (5) applying and drying the siRNA on a solid surface
so that it can be solubilized or suspended in an appropriate
aqueous medium; (6) selecting an appropriate mode of transfection;
(7) selecting a polynucleotide carrier for siRNA such as a lipid;
(8) solubilizing or suspending an siRNA; (9) complexing the siRNA
with the polynucleotide carrier to form an siRNA-carrier complex;
and (10) combining the siRNA-carrier complex with the cell type or
types of choice. Thus, optimizing siRNA RTF protocols can result in
a dramatic improvement over previous forward and reverse
transfection procedures.
[0086] In one embodiment, the present invention can include siRNA
RTF test protocols to implement along with the foregoing
optimizations, which can include any of the following: (a) applying
at least one control siRNA to two or more wells of a multi-well
plate; (b) drying the control siRNA on the floor of each well; (c)
adding, an aqueous medium such as a media or buffer to the control
siRNA in each well in order to solubilize or suspend the control
siRNA, and optionally the solution includes a polynucleotide
carrier so that a control complex can form; (d) adding an
appropriate number of cells to each well in which the control siRNA
is already in solution alone or as an control complex; and (e)
after cells have been added, maintaining the plate under conditions
in which transfection of the cells by the control siRNA can occur.
Following transfection, the cells are subjected to conditions, such
as liquid media, temperature, gas partial pressures, and the like,
in which cell growth and/or cell division will occur and gene
silencing may occur. These conditions can be, but not necessarily,
the same as the conditions under which transfection occurs, and are
well known in the art.
III. Well Plates
[0087] In one embodiment, the present invention includes the use of
control solutions dried in the bottom of a well in a well plate.
The well plates used in connection with the present invention are
preferably formatted and distinct well arrays (e.g., a 48, 96, 384,
or 1536 well plate) that can be purchased from any number of
commercial sources of cell culture plates and other cell culture
surface-containing devices.
[0088] A well plate can be made of glass, polystyrene, other
polymeric material or any equivalent materials, and can form a
rounded or generally flat horizontal bottom having various
generally planar shapes. Additionally, wells having substantially
flat floors can provide uniform cell spacing and monolayer
formation and are preferred. Additionally, it can be preferable for
each plate to have between 32 and 2000 wells, and more preferably
having 1536 wells, 384 wells, or 96 wells; however, a plate having
any number of wells can be used. Also, it can be preferable for the
wells to have a volume that varies between about 5 to about 200
microliters ("uL"), and the total culture area, which is
represented by the well floor, to range between about 0.02 cm.sup.2
and about 0.35 cm.sup.2. Additionally, the wells may not be
modified by any chemical coating, or they can be coated with
poly-L-lysine ("PLL"), laminin, collagen, or equivalent substances
that improve the adherence of cells. Additional descriptions of
well plates can be reviewed in the cross-referenced application
having Attorney Docket No. 16542.1.1, entitled APPARATUS AND SYSTEM
HAVING DRY GENE SILENCING COMPOSITIONS, with Barbara Robertson,
Ph.D., et al. as inventors, which is incorporated herein by
reference.
[0089] Additionally, any of the plates can be included in systems
or kits in accordance with the present invention. Such kits can
include the plates having control compositions and can be
distributed with siRNA solubilizing or suspending solutions,
polynucleotide carriers, carrier solutions, reagents, cell media,
and the like.
IV. Reverse Transfection Testing Plates
[0090] In one embodiment of the present invention, a well plate in
accordance with the foregoing can be configured as a reverse
transfection testing plate ("RTF testing plate"). Accordingly, the
RTF testing plate can include a control composition in one or more
wells. The control composition includes at least a first control
siRNA that can provide an indication of the efficacy of gene
silencing. Optionally, the control composition can have a pool of
control siRNAs. Well plates can be RTF testing plates by having a
control siRNA-containing solution applied to at least one well,
which is then dried in a manner that removes the solution and
leaves a dried control composition.
[0091] In some instances the control siRNA can be solubilized in
one of several types of solutions prior to applying, depositing,
and/or spotting the control siRNA solution onto the well floor, and
drying the material on the plate. The control siRNA can be
dissolved in distilled water that has been treated by one of any
number of art-recognized techniques to eliminate contamination by
RNases such as by ultrafiltration. Alternatively, the control siRNA
may be dissolved in one of several physiologically compatible,
RNase-free buffers, including but not limited to phosphate buffer,
Hanks BSS, Earl's BSS, or physiological saline. These solutions may
contain one or more additional reagents that enhance the stability
of the control siRNA (e.g., RNase inhibitors) or alter the
viscosity of the solution to enhance spotting or drying efficiency
(e.g., sucrose) without changing the properties of the control
siRNA or injuring the cells that are added at subsequent stages in
the RTF testing protocol.
[0092] In still other cases, the control siRNA may be solubilized
in a solution or medium that will enhance spotting, drying, or
sticking to the plate of choice. Optionally, volatile solvents can
be used that are compatible with control siRNA. One example
includes the use of alcohols, such as ethanol, which can be mixed
with water in order to form a volatile solvent that can be readily
dried and leave a dry control composition on the well floor. In
some instances the solution of control siRNA does not contain
lipids that are easily oxidized over the course of time or can be
toxic to cells. In other instances the control siRNA is
pre-complexed with a polynucleotide carrier in a solution before
being deposited and dried to the well floor.
[0093] Accordingly, a predefined amount of control siRNA can be
administered to the well so that when it is dried and then
resuspended, a known amount or concentration of control siRNA is
available for testing gene silencing. The volume of solutions that
are deposited on the bottom of each well can depend upon the
concentration of the stock solution, functionality of the control
siRNA, and desired amount or concentration of control siRNA
available for testing gene silencing. For example, the
concentration of siRNA during transfection can range from picomolar
(e.g., 300-900 pM) for highly functional siRNA (e.g., silence
>90% of target expression at 50-100 nM), to nanomolar (e.g., 100
nM) for siRNA of intermediate functionality (e.g., 70-90% silencing
of target expression at 50-100 nM), and to micromolar (e.g., 1 uM)
for low functionality. For example, for a 96-well plate, deposition
of 5-50 uL of a 1 uM siRNA-containing solution is sufficient to
generate an acceptable concentration of control siRNA for RTF
testing protocols. For smaller or larger sized wells, volumes and
amounts of control siRNA would be adjusted to compensate for the
final concentration of lipid-media/buffer and media that can be
accommodated in each well.
[0094] In one embodiment, the total amount of siRNA in the control
composition can be present in an amount for transfecting cells in
only the well in which it is contained. As such, the total
concentration of control siRNA can be less than about 100 nM when
solubilized or suspended in the aqueous medium during RTF, more
preferably less than about 50 nM, even more preferably the total
concentration of siRNA can be less than about 25 nM, and most
preferably less than about 10 nM when solubilized or suspended in
the aqueous medium during RTF. In another option, the total
concentration of control siRNA can be less than about 1 nM when
solubilized or suspended in the aqueous medium during RTF. For
example, the amount of control siRNA in a 96-well plate can be from
0.1 picomoles ("pm") to about 100 pm, more preferably about 1 pm to
about 75 pm, and most preferably about 10 pm to about 62.5 pm per
well, where corresponding amounts of control siRNA can be
calculated for plates having other numbers of wells.
[0095] Additionally, the amount of control siRNA added to each well
can be sufficient for use in a single RTF testing protocol within
that well. That is, the control siRNA in the control composition
can be present in an amount to only be used with the cells added to
the well. As such, the amount of control siRNA dried in the well
can be insufficient for performing two RTF protocols in two
different wells. This is because the amount of control siRNA
provided in the control composition is configured for a single RTF
testing protocol in order to produce optimal results. Also, this
eliminates the need to make a stock siRNA solution that is
transferred into multiple wells, thereby reducing the complexity of
the RTF testing protocol and increasing efficacy.
[0096] The control siRNA-containing solutions can be deposited into
wells using various well known techniques in the art for depositing
liquids into wells of well plates, which can include manual and
automated processes. Various methods can be used to dry the control
siRNA-containing solution into a control composition. In one
embodiment, the plates are allowed to dry at room temperature in a
sterile setting which allows the deposition solution to evaporate
leaving behind the control siRNA and any other conditioning
compounds, such as salts, sugars, and the like. Dried plates are
preferably vacuum-sealed or sealed in the presence of inert gases
within a sterile container, and stored at temperatures ranging from
-80.degree. C. to 37.degree. C. for extended periods of time
without loss of silencing functionality. Thus, the plates having
the dry control compositions in at least one well can be stored at
room temperature and shipped via traditional routes and still
maintain the integrity and functionality of the control siRNA.
[0097] In one embodiment, the plate can further include a blank
well devoid or substantially devoid of siRNA. That is, the blank
well does not have enough siRNA to provide meaningful gene
silencing. The blank well can be used in concert with control siRNA
to show the effects of the gene silencing condition in the absence
of any siRNA. This can be especially beneficial when assessing the
toxicity of test or control siRNA, or measuring the amount of a
particular mRNA or protein present in a cell. Also, this can
provide data regarding the effect of the polynucleotide carrier on
the cell in the absence of siRNA.
[0098] In one embodiment, the plate can further comprise at least
one test well that has a substantially dry gene silencing
composition. The gene silencing composition can have at least a
first test siRNA which silences a test target gene. Additionally,
the gene silencing composition can be configured such that the test
siRNA is capable of being solubilized or suspended in an aqueous
medium in an amount sufficient for transfecting cells in the well.
The gene silencing composition can be characterized by at least one
of the following: (a) the test siRNA can be unmodified; (b) the
test siRNA can have a stabilizing modification;(c) the test siRNA
can have a modification to limit off-targeting; (d) the test siRNA
can have a conjugate; or (e) the test siRNA can have a hairpin
structure.
V. Control siRNA
[0099] In one embodiment, the dry control compositions include at
least a first control siRNA which can provide an indication of the
efficacy of gene silencing. Optionally, the dry control composition
can include a pool of control siRNAs. The control composition is
configured such that the control siRNA is capable of being
solubilized or suspended in an aqueous medium in an amount
sufficient for transfecting cells in the well. Optionally, the
total amount of control siRNA in the well is sufficient for
implementing reverse transfection only for that well. Additionally,
it is optional for the control siRNA to have at least one of a
modification or a conjugate. Also, the control siRNA can be
rationally designed to target a control gene. Furthermore, the
control composition can include a pool of control siRNAs. Examples
of control siRNA include transfection control siRNA, positive
control siRNA, and negative control siRNA.
[0100] The ability to use control siRNA in a control well to test
the efficacy of gene silencing in other test wells is based on the
consistency between RTF protocols and conditions. In part,
consistency between conditions of a control well and a test well
may enable the results of the control well to provide meaningful
results with regard to the test well. Consistency between the
control well and test well can include consistency in cell type,
cell seeding number, cell density, mode of transfection,
polynucleotide carrier type, polynucleotide carrier concentration,
siRNA concentration, cell culture media, any environmental
conditions to which the both wells are subjected, and the like. In
the instance in which a number of factors are different between the
control well and the test well, the results obtained from the
control well may not be indicative of the gene silencing efficacy
of the test siRNA. Thus, it can be beneficial for the control well
and test well to be on the same plate, or run in concert with
conditions and protocols that are as similar as possible.
[0101] Often, control siRNA are utilized to determine whether or
not the protocol or conditions associated with RTF protocol can
induce non-specific gene silencing or, in some instances, a unique
phenotype. The results obtained from using control siRNA can then
be compared to the test data obtained from test siRNA, wherein the
test siRNA are the siRNA that are being studied in the gene
silencing experiments. Additionally, the control siRNA can be
characterized by the following: (a) the control siRNA can be
unmodified; (b) the control siRNA can have a stabilizing
modification; (c) the control siRNA can have a modification to
limit off-targeting; (d) the control siRNA can have a conjugate; or
(e) the control siRNA can have a hairpin structure.
A. Transfection Control siRNA
[0102] Transfection control siRNA are configured to enable the
level or percentage of cells that have been transfected during an
RTF testing protocol to be identified and/or quantified. This
includes control siRNA that can be used to monitor the efficiency
of an RTF procedure and/or condition. The level of transfection or
percentage of cells that have been transfected can be measured by a
variety of methods, which include identifying the presence of
transfected control siRNA in a cell or measuring the effects of the
control siRNA on the cell. When the effects of control siRNA are
measured, the effects can be direct or indirect, but are usually
well known and reproducible effects that are specifically caused by
the control siRNA. In any event, transfection control siRNA are
used to monitor the efficacy of an RTF protocol and/or condition by
comparing the resulting effects in the control well with
established and reproducible effects that correspond with the
specific transfection control siRNA. This provides an indication of
the transfection and/or gene silencing efficiency.
[0103] In the instance where the presence of the transfection
control siRNA in a transfected cell is identified or quantified
during an RTF testing protocol, the control siRNA can be a type
that is detected without regard to any functionality. That is, the
control siRNA can be configured to be detected in a cell without
measuring any effect that may have been induced. Such transfection
control siRNA can include a label that can be directly measured by
techniques well known in the art. Examples of labels can include
calorimetric labels, fluorescent labels, luminescent labels,
chemiluminescent labels, enzymatic labels, mass labels, radioactive
labels, and the like. Thus, the presence of the control siRNA in a
cell can be measured within the contents of the cell being retained
within the well, or the contents can be removed for analysis. Any
untransfected siRNA can be removed from the cell culture by well
known washing protocols.
[0104] In one embodiment, the transfection control siRNA includes a
colorimetric label. A colorimetric label can be a chromophore that
is detectible by measuring and analyzing the absorbance or
transmitted color spectrum or single wavelength of a sample. As
such a chromophore can be coupled to the siRNA so that the
transfection can be detected or measured by the color spectrum or
single wavelength that is transmitted in response to incident light
or measuring the absorbance of the sample. This can also be
compared to the color spectrum that can be obtained from cells in a
blank well. Alternatively, a colorimetric label can be an enzyme
that reacts with a specific substrate to generate a chromophore
product. The label can be measured and/or quantified by measuring
the amount of light absorbed or transmitted by the product at a
specific wavelength or spectrum. For example, enzymes that can be
used as labels include alkaline phosphatase with para-nitrophenyl
phosphate as the substrate, horseradish peroxidase with hydrogen
peroxide/coupler as the substrate, .beta.-galactosidase with
O-nitrophenylgalactoside as substrate and the like.
[0105] In one embodiment, the transfection control siRNA includes a
fluorescent label. The fluorescent label can be used in order to
photometrically monitor the delivery of the control siRNA into a
cell. Preferably, the fluorescent label is a rhodamine or a
fluorescing however, other fluorescent molecules that can be
coupled with an siRNA can be used. Specific examples of fluorescent
labels include Cy3.TM., Cy5.TM. (Amersham), other cyanine
derivatives, FITC, one of the ALEXA.TM. or BODIPY.TM. dyes
(Molecular Probes, Eugene, Oreg.), a dabsyl moiety, and the like.
It is also possible to use fluorescent microparticles, such as
inorganic fluorescent particles as long as the particle has a size
that does not affect transfection efficiencies. The labels may be
used to visualize the distribution of the labeled siRNA within a
transfected cell. In addition, the label can be used to distinguish
between transfected cells from non-transfected cells. As such, a
population of cells can be transfected with the labeled siRNA and
sorted by FACS. Moreover, the fluorescent labels can be
particularly well suited for HCS and HTC analytical techniques. For
example, cells that have been transfected can be identified, and
then be further examined using HCS analysis.
[0106] In one embodiment, the label can be a luminescent moiety
other than a fluorescent label. Such luminescent moieties can
include phosphorescent microparticles that can be coupled to siRNA.
Preferably, the size of the phosphorescent microparticle does not
affect transfection. Alternatively, the luminescent label can be a
chemiluminescent moiety. A chemiluminescent label can produce light
by a chemical or electrochemical reaction. Chemiluminescence
usually involves the oxidation of an organic compound, such as
luminol or acridinium esters, by an oxidant like hydrogen peroxide
or hypochlorite. Also, chemiluminescent reactions can occur in the
presence of catalysts such as alkaline phosphatase, horseradish
peroxidase, metal ions or metal complexes. As such, a control siRNA
can be labeled with a chemiluminescent organic compound for
transfection, wherein the transfection efficacy is measured by
chemiluminescence after the catalyst is added to the contents of
the transfected cell.
[0107] The use of labeled nucleotides is well known to persons of
ordinary skill, and labels such as enzymatic, mass, or radioactive
labels, may be used in applications in which such types of labels
would be advantageous. Further descriptions of labels that are
applicable for transfection control siRNA in RTF testing protocols
are found in U.S. Provisional Patent Application No. 60/542,646,
60/543,640, and 60/572,270 and International Application
PCT/US04/10343, wherein each is incorporated herein by
reference.
[0108] The label can be attached directly to the control siRNA or
through a linker. The label can be attached to any sense or
antisense nucleotide within the siRNA, but it can be preferable for
the coupling to be through the 3' terminal nucleotide and/or 5'
terminal nucleotide. An internal label may be attached directly or
indirectly through a linker to a nucleotide at a 2' position of the
ribose group, or to another suitable position. For example, the
label can be coupled to a 5-aminoallyl uridine.
[0109] For example, linkers can comprise modified or unmodified
nucleotides, nucleosides, polymers, sugars, carbohydrates,
polyalkylenes such as polyethylene glycols and polypropylene
glycols, polyalcohols, polypropylenes, mixtures of ethylene and
propylene glycols, polyalkylamines, polyamines such as polylysine
and spermidine, polyesters such as poly(ethyl acrylate),
polyphosphodiesters, aliphatics, and alkylenes. An example of a
conjugate and its linker is cholesterol-TEG-phosphoramidite,
wherein the cholesterol is the conjugate and the tetraethylene
glycol ("TEG") and phosphate serve as linkers.
[0110] In one embodiment, the transfection control siRNA can be a
type where its presence is identified or measured by the effects on
a cell. The control siRNA can be detected by an established and
reproducible effect that is caused by the presence of the control
siRNA in a cell. For example, the control siRNA can be a type that
causes cell toxicity and/or death when transfected into a cell, or
can cause an identifiable or measurable response not related to
RNAi. Cytotoxic siRNA can be used as transfection control siRNA
because the amount of cell toxicity can be directly related to the
siRNA. The gene silencing efficacy of a test well can be identified
by the amount of toxicity induced by the cytotoxic siRNA in the
control well. Also, the effect of the cytotoxic siRNA can be
compared to cells in blank wells as well as to cells in test wells.
In the instance the cytotoxic siRNA induces cell death and the
cells in the blank wells or test wells do not show any toxic
effects, the transfection and/or gene silencing efficacy of the
test well can be compared to level of cell death induced by the
cytotoxic siRNA. However, established and reproducible toxic
effects that arise from cytotoxic siRNA can be analyzed without
being compared to cells in other wells.
[0111] In one embodiment, the transfection control siRNA is a
cytotoxic siRNA. Some cytotoxic siRNA which have been identified
include sense polynucleotide sequences that include the following
motifs: AAA; UUU; GCCA; or UGGC. Also, an siRNA can be toxic by
including a long polynucleotide sequence. The toxic control siRNA
can include a long dsRNA that is about 50 base pairs, and
preferably longer than 50 base pairs. Optionally, the toxic control
siRNA can induce an interferon response that is toxic to the cells.
Cytotoxic transfection control siRNA can be represented by
Dharmacon's siCONTROL.TM. TOX. Additionally, siRNA that induce a
toxic response through the RNAi pathway by inhibiting production of
a protein vital to cell viability can be used as cytotoxic
siRNA.
[0112] B. Positive Control siRNA
[0113] Positive control siRNA can be well characterized siRNA that
silence a well known gene. That is, the control siRNA silence a
known gene with established and reproducible results. As such, the
positive control siRNA can provide meaningful data regarding the
efficacy of transfection and/or gene silencing in a test plate
having a similar RTF protocol or conditions. In part, this is
because the control siRNA are known to systematically silence a
known gene to stop production of a known protein, wherein the known
gene and known protein can be referred to as a control gene and
control protein or control polypeptide, respectively.
[0114] Positive control siRNA can be distinguished from test siRNA
by a number of characteristics. Usually, test siRNA are being
tested to identify whether or not a target test gene will be
silenced in an RTF protocol or condition. On the other hand,
positive control siRNA have well known sequences and/or
characteristics that systematically silence a well known gene in a
reproducible and measurable manner. This allows the positive
control siRNA to provide meaningful results regarding the efficacy
of the test siRNA because when the positive control gene is not
silenced or is overly silenced in the control well, the results
obtained from a test well from using the test siRNA may be
unreliable. Examples of positive control siRNA include siRNA that
can silence MAP kinase genes, glyceraldehyde-3-phosphate
dehydrogenase ("GAPDH") gene, cyclophilin B ("cyclo") gene, Lamin
A/C genes, and other well established genes that can be silenced
with siRNA having specific polynucleotide sequences. Specific
examples of positive control siRNA include Dharmacon's
siCONTROL.TM. GAPD, siCONTROL.TM. Cyclophilin B, and siCONTROL.TM.
Lamin A/C.
[0115] Positive control siRNA can also include siRNA that inhibit
the RNAi pathway, which uses siRNA targeting RISC genes, Ago2
genes, eIF2C2 genes, and the like for silencing. Additionally,
these positive control siRNA can silence human, mouse, rat, or
other animal control genes. In addition, positive control siRNA can
target any gene that when silenced, gives a predictable result in
e.g. a phenotypic
[0116] In one embodiment, the positive control siRNA can be
selected to optimize functionality in silencing the control gene.
Preferably, the positive control siRNA has between 50% and 100%
gene silencing functionality with respect to the control gene, more
preferably between 70% and 100%, even more preferably between 80%
and 100%, and most preferably between 90% and 100% functionality in
silencing the control gene.
[0117] Additionally, the positive control siRNA antisense strand
can have varying levels of complementarity with the control
sequence to which it targets (e.g., control mRNA or gene).
Preferably, the antisense strand can have about 50-100%
complementarity with the target sequence, more preferably, about
70-100% complementarity, even more preferably about 80-100%
complementarity, still even more preferably about 90-100%
complementarity, and most preferably about 100% complementarity
with the control sequence.
[0118] In one aspect, the positive control siRNA can be selected by
rational design. As such, the positive control siRNA is selected
using one or more formulas that identify sequences that have
desirable attributes and are more highly functional. Highly
functional positive control siRNA can be identified by rational
design in order to perform more consistently and reproducibly than
less functional siRNA under a wide range of conditions found in RTF
formats (e.g., cell densities).
[0119] In one embodiment, it can be preferable to select positive
control siRNA that have been previously identified from lists of
siRNA that have been selected using rational design algorithms. As
such, the control siRNA can be selected from Table I of the
incorporated provisional application having Ser. No. 60/678,165.
Table I is entitled "siGENOME Sequences for Human siRNA," and
consists of columns "Gene Name," "Accession No.," "Sequence," and
"SEQ. ID NO." Table I lists 92,448 19-mer siRNA sense strand
sequences, where antisense strand sequences were omitted for
clarity. The siRNA sequences listed in Table I includes SEQ. ID
NOs. 1-92,448, wherein each preferably can also include a 3' UU
overhang on the sense strand and/or on the antisense strand. Each
of the 92,448 sequences of Table I, when used in an siRNA, can also
comprise a 5' phosphate on the antisense strand. Of the 92,448
sequences listed in Table I, 19,559 have an on-targeting set of
modifications. A list of sequences, identified by SEQ. ID NO., that
have on-target modifications is presented in Table II, entitled
"List of Table I Sequences Having On-Target Modifications
Identified by SEQ. ID NO." On-target modifications are on SEQ. ID
NOs. 1-22,300.
[0120] C. Negative Control siRNA
[0121] Negative control siRNA can be characterized as not being
functional within the RNAi pathway, and thereby do not induce gene
silencing. This can be accomplished by various mechanisms which
include siRNA that are not functional, siRNA that inhibit uptake
and processing by RISC, and/or siRNA that do not have
complementarity to a gene or mRNA. For example, the negative
control siRNA can fail to enter RISC. As such, negative control
siRNA can provide meaningful results that are well known and
reproducible by not affecting the cellular processes of transfected
cells. Also, negative control siRNA can provide indications of gene
silencing conditions and protocols by testing for any induced
toxicity, loss of cellular function, or non-specific inhibition of
protein production.
[0122] In one embodiment, the negative control siRNA is
non-functional siRNA, which does not function in the RNAi pathway.
The non-functional siRNA can provide an indication of transfection
efficacy by monitoring the response of the transfected cell.
Instances in which non-functional siRNA cause changes in gene
expression, cell function, or phenotype may be the result of
factors other than RNAi mediated gene silencing, and can provide an
indication regarding the efficacy of gene silencing of test wells
having substantially similar RTF protocols and/or conditions.
Non-functional siRNA can have certain sequences and/or chemical
modifications in order to induce non-functionality. Additionally,
non-functional siRNA can have a 17 base pair duplex or any duplex
having less than 18 base pairs. For example, non-functional siRNA
can include a 17 base pair duplex with 2' modifications at the
first and second sense nucleotides and on the first and second
antisense nucleotides. In another example, a non-functional siRNA
can include a 19 base pair duplex with 5' deoxynucleotides on the
5' end of the sense and the antisense strands.
[0123] In one embodiment, the negative control siRNA includes
sequence and/or modifications that inhibit uptake and processing by
RISC. A negative control siRNA can include a modification or size
that inhibits being taken in and processed by RISC. That is, RISC
is not able to perform a function with mRNA having the sequence of
the negative control siRNA, wherein the negative control has a
modification or size that prevents being taken into RISC. This can
be used to modify siRNA having selected sequences that can be used
for comparative purposes with test siRNA with the same sequence but
not having the modifications. As such, any gene silencing that
arises from the negative control siRNA may result from non-specific
silencing and provide an indication that the data obtained from the
test siRNA is not reliable. An example of the negative control
siRNA that are not taken in or processed by RISC can include
siCONTRO.TM. RISC-Free (Dharmacon, Inc.).
[0124] For example, the negative control siRNA that are
non-functional or are not taken in and processed by RISC can have
various 2' modifications. This can include control siRNA that
contain 2' modifications on the first and second sense nucleotides,
2' modifications on at least one through all pyrimidine sense
nucleotides, 2' modifications on the first and/or second antisense
nucleotides, 2' modifications on at least one through all
pyrimidine antisense nucleotides, and/or a 5' carbon having a
phosphate modification at the sense or antisense 5' terminal
nucleotide. The 2' modifications can be 2'-O-aliphatic
modifications or 2'-halogen modifications. The control siRNA can
also include internucleotide modifications with phosphorothioates
or methylphosphonates.
[0125] The 2' modifications can be characterized by as
2'-O-aliphatic modifications such as 2'-O-alkyl modifications. For
example, the 2'-O-alkyl can be selected from the group consisting
of 2'-O-methyl, 2'-O-ethyl, 2'-O-propyl, 2'-O-isopropyl,
2'-O-butyl, 2'-O-isobutyl, 2'-O-ethyl-O-methyl (i.e.,
--CH.sub.2CH.sub.2OCH.sub.3), 2'-O-ethyl-OH (i.e.,
--OCH.sub.2CH.sub.2OH), 2'-orthoester, 2'-ACE group orthoester, and
combinations thereof. Most preferably, the 2'-O-alkyl modification
is a 2'-O-methyl moiety. Additionally, the 2'-halogen modifications
can be selected from the group consisting fluorine, chlorine,
bromine, or iodine; however, fluorine is preferred.
[0126] In one embodiment, the negative control siRNA can be an
siRNA that has a sense and/or antisense strand with limited or
non-functional complementarity to a gene or mRNA sequence. This can
include siRNA that are bioinformatically designed to minimize any
potential targeting of any known human or animal gene. The
non-targeting negative control siRNA can modified or unmodified,
and examples include Dharmacon's siCONTRO.TM. Non-Targeting siRNA
#1 and siCONTROL.TM. Non-Targeting pool.
[0127] D. Dual Function Control siRNA
[0128] In one embodiment, the present invention includes dual
function control siRNA. A dual function control siRNA can be used
for two different control studies. This can include a single
control siRNA that functions both as a transfection control and a
positive control. Also, this can include a single control siRNA
that functions both as a transfection control and a negative
control. Examples of dual function controls include any positive or
negative control that includes a label, such as a fluorescent label
(e.g., siGLO.TM. Cyclophilin B, siGLO.TM. Lamin A/C, siGLO.TM.
RISC-Free, each from Dharmacon, Inc.).
[0129] E. Control siRNA Configurations
[0130] The control siRNA may be used individually (e.g., one siRNA
sequence per well) or as part of a pool. A pool of control siRNAs,
as defined herein, refers to the use of at least two different
control siRNAs within a specific well, and usually at least four
different control siRNAs. Each control siRNA can include between 18
and 31 base pairs, more preferably between 19 and 26 base pairs,
and most preferably 19 and 21 base pairs. However, toxic control
siRNA can have duplex regions with about 50 base pairs or greater
than 50 base pairs, and some non-functional control siRNA can
include less than 18 base pairs. Each control siRNA can include a
sense strand and an antisense strand, which are preferably at least
substantially complementary to each other over the range of the
duplex region. It is most preferable for the duplex region to have
about 100% complementarity.
[0131] Additionally, the control siRNA can have overhangs, bulges,
mismatches, stability modifications, specificity modifications,
hairpin structures, or other common features on target siRNA.
Accordingly, additional information regarding these features can be
reviewed in the cross-referenced patent application filed herewith
having Attorney Docket No. 16542.1.1, entitled APPARATUS AND SYSTEM
HAVING DRY GENE SILENCING COMPOSITIONS, with Barbara Robertson,
Ph.D., et al. as inventors.
[0132] A reduction in off-targeting or increased specificity can
also be achieved by using control siRNA concentrations that are
below the level that induces off-target effects. As an example,
transfection of a single control siRNA at 100 nM can induce 90%
silencing, yet the high concentration of the siRNA may also induce
off-target effects. In contrast, a pool of four control siRNAs
(e.g., total concentration of 100 nM, 25 nM each) can similarly
induce 90% silencing. Since each siRNA is at a four-fold lower
concentration, the total number of off-targets is fewer. Thus, in
order to obtain control gene silencing with inhibited or no
off-target effects, a highly functional siRNA can be used at low
concentrations, or pools of control siRNA targeting the same gene
can be used with each siRNA of the control pool having a
concentration that is sufficiently low to minimize off-target
effects. Preferably, the total amount of control siRNAs can be
delivered at concentrations that are less than or equal to about
100 nM, more preferably less than or equal to about 50 nM, even
more preferably less than or equal to about 25 nM, and most
preferably less than or equal to about 10 nM.
VII. Polynucleotide Carriers
[0133] In one embodiment, the present invention includes
polynucleotide carriers that can interact with a control siRNA, and
transport the control siRNA across a cell membrane. However, in
other embodiments of the invention modes of transfection can be
implemented without carriers, such as by electrophoresis,
precipitation, particle bombardment, optoporation, and
microinjection. Usually, polynucleotide carriers include a positive
charge that interacts with the negatively charged phosphates on the
polynucleotide backbone. Polynucleotide carriers are well known in
the art of cellular nucleic acid deliver. Preferred polynucleotide
carriers include polymers, lipids, lipopolymers, lipid-peptide
mixtures, and the like that are capable of complexing with an siRNA
and delivering the siRNA into a cell in a manner that retains the
gene silencing functionality without being overly toxic. As such,
routine experimentation can be implemented with procedures
described herein with respect to optimizing RTF protocols in order
to identify the optimal polynucleotide carrier for a certain system
or cell.
[0134] In one embodiment, lipids or lipid-peptide mixtures are
preferable for introducing siRNA into a target cell. Typically, the
lipid is a cationic lipid. Cationic lipids that can be used to
introduce siRNA into cells can be characterized by having little or
no toxicity (e.g., defined as less than 15-20% toxicity), which can
be measured by AlamarBlue or equivalent cell viability assays.
However, not all lipids are functionally equivalent and certain
lipids can perform better with specific cell lines. Thus, the
foregoing optimization procedures can be employed to determine an
appropriate lipid and lipid concentration for delivering siRNA for
a specific cell line. Peptides that have affinity to one or more
proteins, lipids, lipid-polysaccharide, or other components of the
cell membrane can be conjugated to the siRNA and used independent
of lipids or advantageously combined with one or more lipids to
form a polynucleotide carrier. Such lipid-peptide mixtures can
enhance RTF of siRNA. Cholesterol conjugates can be similarly
coupled to the siRNA and be used independent of polynucleotide
carriers or advantageously combined therewith.
[0135] Briefly, in order to identify whether a given lipid is
acceptable for siRNA RTF testing protocols, two or more well
characterized control siRNAs can be tested under a variety of
lipid, media, and siRNA concentrations using the optimizing
protocols described herein. Subsequently, the level of transfection
and/or gene silencing and/or the level of cell death are quantified
using art-accepted techniques. Suitable lipids for siRNA RTF
testing protocols include OLIGOFECTAMINE.TM., TransIT-TKO.TM., or
TBIO Lipid 6.TM., LIPOFECTAMINE.TM. 2000, lipids DharmaFECT.TM. 1,
DharmaFECT.TM. 2, DharmaFECT.TM. 3, and DharmaFECT.TM. 4
(Dharmacon, Inc.). The term "DharmaFECT.TM." (followed by any of
the numerals 1, 2, 3, or 4) or the phrase "DharmaFECT.TM.
transfection reagent," refers to one or more lipid-based
transfection reagents that have been optimized to transfect siRNA
rather than larger nucleic acids (e.g., plasmids). Additional
information on lipids can be obtained in U.S. Pat. Nos. 5,674,108,
5,834,439, 6,110,916, 6,399,663, and 6,716,582, and international
publications WO 00/12454 and WO 97/42819, wherein each is
incorporated herein by reference.
[0136] The formation of a functional control siRNA-lipid complex
can be prepared by combining control siRNA and the lipid. As such,
an appropriate volume of lipid at a selected concentration can be
combined with a volume of media and/or buffer to form a lipid-media
or lipid-buffer having a suitable concentration of lipid. For
example, a volume of lipid media ranging from about 5-50 microliter
("uL") can include about 0.03-2 micrograms ("ug") of lipid to be
introduced into each well of a 96-well plate, and the amount of
lipid can be changed to correspond with other well sizes. The
choice of media and/or buffer can improve the efficiency of the RTF
protocol. Some media contain one or more additives that induce cell
toxicity and/or non-specific gene modulation during RTF testing
protocols. Examples of preferred media or buffers include
Opti-MEM.TM. (GIBCO, Cat. #31985-070), HyQ-MEM-RS.TM. (HyClone,
Cat.#SH30564.01), Hanks Balanced Salt Solution.TM., or equivalent
media. A suitable media can be identified by employing the
optimization protocol described herein.
[0137] The lipid-media or lipid-buffer can be introduced into a
well by a variety of methods including hand-held single and
multi-channel pipettes, or more advanced and automated delivery
systems that can inject measured volumes of the lipid solution into
a well. The lipid solution can be incubated in the well that
contains the dried control composition for a period of time that is
sufficient to solubilize or suspend the siRNA, and to form control
complexes. In general, the process of siRNA control solubilization
and complex formation can require about 20 minutes, but usually not
more than 120 minutes. The complex formation process is generally
performed at room temperature, but can be performed at temperatures
ranging from 4-37.degree. C. In some instances, the lipid and
control siRNA can be mixed by agitating the plate (e.g., swirl,
vortex, sonicate) for brief periods (e.g.; seconds--minutes) to
enhance the rate of control siRNA solubilization and complex
formation.
[0138] Additionally, any of the foregoing polynucleotide carriers
can be included in systems or kits in accordance with the present
invention. Such kits can include the plates having control
compositions, and can be distributed with siRNA solubilizing or
suspending solutions, polynucleotide carriers, carrier solutions,
reagents, cell media, and the like.
VIII. Well Arrangements
[0139] In one embodiment, the siRNA RTF testing plates that include
multiple wells having different dry control compositions can have
the wells organized into predefined arrangements. Such arrangements
can correspond to the type of assay being employed with the siRNA
RTF testing plate. That is, when a family of genes is being
studied, a first control siRNA can be organized in one column or
row while a second control or targeting siRNA can be organized in a
different column or row. Thus, the wells can be organized into a
pre-selected arrangement so that particular control siRNAs are in a
pre-selected pattern on a plate. The pre-selected pattern can
include various control wells, such as those that include one or
more negative, positive, and/or transfection controls. Also, the
pre-selected pattern can include wells that are empty or
substantially devoid of any siRNA, which can be used as controls
and for calibrations.
[0140] It can be beneficial to have control siRNAs that are
pre-dried in corresponding wells of different well plates so that
multiple RTF testing plates can be prepared simultaneously. This
can allow for RTF testing plates to have gene silencing
compositions at standardized positions and amounts of control
siRNAs, which is beneficial for using standardized well plates in
multiple experiments that can be conducted over time without
introducing variability between the plates. The use of standardized
plate arrangements can provide a series of plates that can be used
over time and provide data that can be analyzed together.
[0141] For example, a plate comprising a plurality of columns of
wells can include a transfection control in the first column,
positive controls for RNAi in the second column, negative controls
for RNAi in a third column, a pool of siRNAs directed against a
single target in a fourth column, and individual members of the
siRNA pool that comprise the fourth column are in subsequent
columns, such as the fifth through twelfth columns. Alternatively,
the fifth through twelfth columns can comprise different
concentrations of each siRNA in the pool of the fourth column, with
the amount of siRNA increasing from well to well or decreasing from
well to well. Each well can include one concentration of each siRNA
in the pool, or two, three, four, five, or more concentrations of
each siRNA in the pool can be in different wells. The number of
concentrations of siRNA that can be used is limited only by the
number of wells on the plate; however, multiple plates can be
configured to be used together with a predefined pattern that
spreads across all the plates. Additionally, the pre-selected
patterns of control siRNA concentration gradients can be used as a
pattern that can be observed so that the optimal amount of each
control siRNA can be determined by observing the level of
transfection and/or gene silencing number of concentrations of that
particular control siRNA.
[0142] FIGS. 1A and 1B illustrate embodiments of plate arrangements
similar with the foregoing concentrations arrangements. While the
wells are shown to be square, it should be recognized that they can
be any shape. Also, the well plate can include any number of wells,
and the number of wells depicted is merely for example. In the
figures the wells are defined as follows: "Tc" indicates a
transfection control well, wherein the increasing corresponding
numbers identify different transfection controls; blank wells
indicate wells devoid or substantially devoid of any siRNA; "+"
indicates a positive control; "-" indicates negative controls; "P1"
through "P1.sub.N" indicate a first pool which silences a first
gene at a concentration gradient; "P2" through "P2.sub.N" indicate
a second pool which silences a second gene at a concentration
gradient; "1A" through "1.sub.N" indicate a first individual test
siRNA of the first pool at a concentration gradient; "2A" through
"2.sub.N" indicate a second individual test siRNA of the first pool
at a concentration gradient; "3A" through "3.sub.N" indicate a
third individual test siRNA of the first pool at a concentration
gradient; "4A" through "4.sub.N" indicate a first individual test
siRNA of the second pool at a concentration gradient; "5A" through
"5.sub.N" indicate a a second individual test siRNA of the second
pool at a concentration gradient; and "6A" through "6.sub.N"
indicate a third individual test siRNA of the second pool at a
concentration gradient. Thus, FIG. 1A illustrates a well plate
assaying a single pool, and FIG. 1B illustrates a well plate
assaying multiple pools. Additionally, a well plate can include
more than two pools.
[0143] FIG. 2A. is a schematic diagram that illustrates an
embodiment of a well plate having control siRNA. More particularly,
wells A1-H1, G2, and H2 are blank wells devoid of siRNA. For
example, wells A1-H1 are available for RTF testing controls. Well
G2 can be used as a mock transfection control, and well H2 can be
an untreated cell control. Wells A2-F2 contain negative and
positive transfection control siRNAs. More particularly, the
control wells include the following: well A2 includes a
non-targeting negative control siRNA (e.g., siCONTROL.TM.
Non-Targeting siRNA pool from Dharmacon, Inc.); well B2 includes an
siRNA that inhibits being taken in and processed by RISC (e.g.,
siCONTRO.TM. RISC-Free from Dharmacon, Inc.); well C2 includes a
dual control siRNA that can be used as a negative control and a
transfection control (e.g., siGLO.TM. RISC-Free from Dharmacon,
Inc.); well D2 includes a positive control siRNA targeting GAPDH
control gene (e.g, siCONTROL.TM. GAPD from Dharmacon, Inc.); well
E2 includes a positive control siRNA targeting cyclophilin B
control gene (e.g., siCONTRO.TM. Cyclophilin B siRNA from
Dharmacon, Inc.); and well F2 is a positive control siRNA targeting
Lamin A/C control gene (e.g., siCONTRO.TM. Lamin A/C from
Dharmacon). The wells in columns 3-12 can each contain a test siRNA
or test siRNA pool such as any of Dharmacon's SMARTpool.TM. siRNA
reagents. Alternatively, well F2 can be a user defined control
siRNA.
[0144] FIG. 2B is a schematic diagram that illustrates an
embodiment of an optimization well plate or an RTF testing plate
having control siRNA. As shown, rows A-C can contain three negative
control siRNAs (e.g., siCONTRO.TM. Non-Targeting siRNA pool,
siCONTROL.TM. RISC-Free siRNA, siGLO.TM. RISC-Free siRNA, each from
Dharmacon, Inc.). Rows D-E can contain three positive transfection
control siRNAs (e.g., siCONTROL.TM. GAPD, siCONTROL.TM. Cyclophilin
B siRNA, siCONTROL.TM. Lamin A/C siRNA, each from Dharmacon, Inc.).
Alternatively, Row F can be a user defined control siRNA. Row G
does not contain siRNA and can be used for mock-transfected cells
in order to study the effect of the transfection reagent alone on
cell viability and/or mRNA expression. Row H does not contain siRNA
and is can be used for untreated cells, and can serve as a 100%
viability control and/or 100% mRNA level control.
[0145] FIG. 2C is a schematic diagram that illustrates an
embodiment of an optimization plate or an RTF testing plate having
control siRNA. The testing plate includes an arrangement of wells
that can be used in a procedure for optimizing RTF conditions. As
shown, column 1 includes blank wells devoid of siRNA, and can be
used as a blank that does not receive cells. This column can be
used for a standard curve or other experimental controls that may
be desired for an RTF testing protocol for assessing the efficacy
of gene silencing. Column 2 includes blank wells devoid of siRNA,
and can be used as a blank that does receive cells. This column can
be used as an untreated reference for different volumes of
polynucleotide carrier, such as DharmaFECT.TM. transfection
reagent, which is tested in columns 3-12. Columns 3-7 can contain
negative control siRNAs, which serve as negative control samples
for each DharmaFECT.TM. transfection reagent volume used. As shown,
up to five volumes of each DharmaFECT.TM. transfection reagent may
be tested in one plate. In each row, the DharmaFECT.TM.
transfection reagent volumes in columns 3-7 can be repeated in
columns 8-12. This can allow the negative control siRNAs to serve
as references for any gene silencing that occurs in the wells of
columns 8-12, which contain positive transfection control siRNAs.
Additionally, rows A-D can be seeded with a low cell density, and
rows E-H can be seeded with a high cell density. Thus, the plate
can include a combination of different RTF conditions, which can be
used to determine the optimal amount of DharmaFECT.TM. transfection
reagent, DharmaFECT.TM. transfection reagent volume, and cell
number.
[0146] FIG. 2D is a schematic diagram that illustrates an
embodiment of an optimization well plate or an RTF testing plate
having control siRNA. As shown, the plate is arranged with blanks,
negative control siRNA, and positive control siRNA as in FIG. 2C.
However, this plate can be assayed with different polynucleotide
carrier concentrations. Rows A-D can be used with low cell
densities with DharmaFECT.TM. transfection reagent at volumes
ranging from 0.03 uL/well (e.g., columns 3 and 8) to 0.5 uL/well
(e.g., columns 7 and 12). Rows E-H can be used with high cell
densities with DharmaFECT.TM. transfection reagent at volumes
ranging from 0.06 uL/well (e.g., columns 3 and 8) to 1.0 uL/well
(e.g., columns 7 and 12).
EXAMPLES
[0147] The following examples are provided to describe some
embodiments of the present invention in a manner that can be use by
one of skill in the art to practice the present invention.
Additionally, the following examples include experiments that were
actually performed as well as prophetic experiments. Additional
examples and supplementary information for the following examples
can be reviewed in the incorporated references having Attorney
Docket No. 16542.1.1, entitled APPARATUS AND SYSTEM HAVING DRY GENE
SILENCING COMPOSITIONS, with Barbara Robertson, Ph.D., et al. as
inventors, Attorney Docket No. 16542.1.2, entitled APPARATUS AND
SYSTEM HAVING DRY GENE SILENCING POOLS, with Barbara Robertson,
Ph.D., et al. as inventors, and U.S. Provisional Application Ser.
No. 60/678,165. The polynucleotide sequences that were used in the
examples can be found in Tables I-IV of U.S. Provisional
Application Ser. No. 60/678,165, and the sequence listing of the
reference having Attorney Docket No. 16542.1.1, entitled APPARATUS
AND SYSTEM HAVING DRY GENE SILENCING COMPOSITIONS, with Barbara
Robertson, Ph.D., et al. as inventors.
Example 1
[0148] The effect of plate conditions on RTF protocols was assayed
in order to determine optimum conditions. The different types of
plate coatings that were studied included untreated,
fibronectin-treated, poly-L-Lysine treated, MATRIGEL.TM.-treated,
and CELLBIND.TM. plates. In the poly-L-lysine plates, each well was
treated with 50 uL of 5 ug/uL poly-L-lysine for 1 hour, and washed
with ddH.sub.2O (3.times.) and dried under a UV light for 20
minutes. MATRIGEL.TM. plates were obtained from BD Biosciences
(Catalog No. 354607, Bedford, Mass.). Fibronectin plates were
purchased from Becton Dickinson Labware (Biocoat cellware, Catalog
No. 354409, Bedford, Mass.) and CELLBIND.TM. plates were obtained
from Corning (Catalog No. 3300). For each study, varying amounts of
human cyclophilin B siRNA (e.g., cyclo3) were added to different
wells at 0-250 nM.
[0149] The results of these studies are provided in FIGS. 3A-3J,
which are graphs illustrating the efficacy of the RTF testing
procedures. The graph of FIG. 3A depicts the cell viability of the
untreated plates, wherein the cell viability drops fairly steadily
as the lipid concentration increases. At low lipid concentrations
(e.g. 0.125 ug lipid per 100 uL) the cells were sufficiently viable
to provide meaningful gene silencing results. The graph of FIG. 3B
depicts the cyclophilin B siRNA successfully silenced the target
gene. The graph of FIG. 3C depicts the cell viability of polylysine
plates to similarly decrease as the lipid concentration increase,
and the lower lipid concentrations were not overly toxic to the
cells. The graph of FIG. 3D depicts the cyclophilin B siRNA
successfully silenced the target gene. The graph in FIG. 3E depicts
the CELLBIND.TM. plates to have reduced toxicity with acceptable
levels of cell viability being preserved up to 0.25 ug of lipid per
100 uL. However, the graph of FIG. 3F shows that gene silencing was
only moderate, and was determined to be unacceptable. The graph in
FIG. 3G depicts the MATRIGEL.TM. plates to have overall poor cell
viability under all conditions, and the graph of FIG. 3H was
considered to be unreliable. The graph of FIG. 31 depicts the
fibronectin-coated plates to also have poor cell viability, and the
graph of FIG. 3J was considered to be unreliable.
Example 2
[0150] The optimization of RTF protocols to induce gene silencing
was studied by assessing the siRNA functionality in relation to
cell density. The siRNAs of varying functionalities (e.g., F50-F95)
were reverse transfected under a range of cell densities (e.g.,
10,000-40,000 cells per well) using lipid concentrations that
induced minimal levels of cell toxicity (0.063 ug of lipid per 100
uL for 10,000 cells per well, 0.125 ug of lipid per 100 uL for
20,000 cells per well, and 0.25 ug of lipid per 100 uL for 40,000
cells per well). The study was performed with control cyclophilin B
siRNA (e.g., cyclo3, cyclo28, and cyclo37 with functionalities of
95, 75, and 50, respectively).
[0151] FIG. 4 is a graphical representation of the gene silencing
of increasing concentrations of siRNA in relation to the increasing
number of cells. The graph shows that highly functional siRNA
induced greater gene silencing under a broad range of conditions.
The cyclo3 siRNA induced 60% or more silencing at all three cell
concentrations. In contrast, less functional molecules (e.g., cyclo
28) performed well at only low cell densities (e.g., 10,000 cells
per well), but less well at higher cell densities (e.g., 20-40,000
cells per well). Thus, increased functional siRNA, which are
rationally designed greatly improve gene silencing.
Example 3
[0152] A population of randomly selected siRNAs derived from a walk
targeting DBI (e.g., NM.sub.--020548, position 202-291) was
assessed for the ability to induce toxicity. The collection of
siRNAs consisted of 90 individual (e.g., 19 bp) duplexes, and
covered the respective regions in single base steps. Duplexes were
forward transfected into HeLa cells using LIPOFECTAMINE.TM. 2000,
and a threshold of 75% cell viability was used as the cutoff to
distinguish toxic from nontoxic sequences.
[0153] FIG. 5A is a graphical representation of the results of the
toxicity study. As shown, the siRNAs transfected under these
conditions were observed to induce varying levels of cellular
toxicity. Overall, 14 out of 90 siRNA duplexes (e.g., 15.5%) were
found to decrease cellular viability below 75%, which is identified
by the horizontal dashed line. These toxic siRNA can be identified
by the numbers within the boxes that show cell survival below the
dashed line, and can be used as transfection control siRNA
[0154] FIG. 5B is a graphical representation of an the cell
toxicity and viability obtained from individual siRNAs of 48
functional (e.g., >70% silencing) pools of four siRNA targeting
12 different genes. Only twelve of the forty-eight sequences (e.g.,
25%) decreased cellular viability below 75%.
[0155] FIG. 5C is a graphical representation of the toxicity of
exemplary siRNA of the pools depicted in FIG. 5B. While all eight
duplexes targeting MAP2K1 and MAP2K2 show greater than 80% gene
silencing, only a single siRNA in each quartet reduces cell
viability below 75% (e.g., MAP2K1-d4 and MAP2K2-d3). Thus, as the
remaining siRNAs in each group were equally functional, but
non-toxic, the toxicity induced by MAP2K1-d4 and MAP2K2-d3 is
unrelated to target knockdown.
[0156] A linear display of the distribution of toxic siRNA along
the DBI walk showed that the dispersal of these toxic sequences was
frequently non-random (i.e., clustered) and suggested the presence
of one or more motifs that were responsible for the observed
toxicity (e.g., see boxed areas of FIG. 5A). Subsequent analysis of
the toxic sequences from the random functional siRNA set revealed
that all twelve sequences contained either an AAA/UUU or GCCA/UGGC
motif. To determine whether a correlation existed between the
motifs and toxicity, three additional, randomly selected, groups of
siRNA that contained either AAA/UUU motifs, GCCA/UGGC motifs, or
neither motif, were chosen and tested for the ability to induce
cell death. FIGS. 6A-6C are graphical representations that shown
the siRNA containing the AAA/UUU and GCCA/UGGC motifs exhibited a
higher probability of inducing toxicity (e.g., 56% and 53%,
respectively) in comparison with the non-motif siRNA (e.g., 6%).
Since the statistical analysis (e.g., T-Test) p-value was
1.3.times.10.sup.-7 for these two samples, the results show a
strong correlation exists between siRNA induced cellular toxicity
and delivery of duplexes containing the AAA/UUU or GCCA/UGGC
motifs. Thus, siRNA having toxic motifs can be used as transfection
controls.
Example 4
[0157] The ability of siRNA having a toxic motif to induce cell
death was studied while the RNAi pathway was severely compromised.
Previous studies revealed that eIF2C2/hAgo2 is required for mRNA
cleavage, and that silencing of these gene products can severely
cripple the RNAi pathway. In a control study shown in FIG. 11A, a
first set of HeLa cells were forward transfected with siRNA
directed against eIF2C2 (e.g., siRNA-eIF2C2), and a second set were
transfected with control siRNA (e.g., siRNA-RISC-Free) that are not
processed by RISC in T1. Each set of the HeLa cells where then
transfected with siRNA-RF (i.e., siRNA-RISC-Free) and pEGFP in
T2.
[0158] The results of the control study of FIG. 7A can be viewed in
the images depicted in FIGS. 7B-7I. The results shown in FIGS. 7B
and 7C show that the siRNA-RISC free does not inhibit EGFP
expression from pEGFP. However, FIGS. 7D and 7E show that the
siRNA-EGFP was able to inhibit production of the green fluorescent
protein when the RNAi pathway was not compromised. The results in
7F and 7G show that the even if siRNA-eIF2C2 inhibited the
functionality of the RNAi pathway, the control siRNA-RF did not
have any effect because the cells expressed the green fluorescent
protein. Additionally, FIGS. 7H and 7I show that the siRNA-eIF2C2
inhibited the functionality of the RNAi pathway because the pEGFP
show the cells were transfected and expressed the green fluorescent
protein in the presence of siRNA-EGFP.
[0159] The importance of the RNAi pathway in siRNA-induced cell
death was tested by transfecting HeLa with the siRNA-eIF2C2 and
control siRNA-RF (T1 of FIG. 7A Experiment). The HeLa cells were
subsequently transfected with toxic siRNA containing either the
AAA/UUU or GCCA/UGG motifs (T2 of FIG. 7A Experiment). FIG. 7J is a
graphical representation of the gene silencing obtained with
siRNA-eIF2C2, which eliminated the ability of toxic siRNA to induce
cell death. As parallel experiments, cells were pre-transfected
with control siRNA-RF (T1) that exhibited toxicity characteristic
of these sequences, and it was concluded that an intact RNAi
pathway was necessary for siRNA-induced toxicity.
Example 5
[0160] Additional experiments were conducted to determine the
involvement of the RNAi pathway in siRNA induced toxicity. The
ability of toxic siRNA to induce cell death was tested with siRNA
having 19 and 17 base pairs. Previous studies have shown that
duplexes that are shorter than 19 base pairs targeted mRNA
sequences inefficiently, which suggests that Dicer and/or RISC fail
to mediate RNAi when duplex sequence length drops to some level
below 19 base pairs.
[0161] FIG. 8 is a graphical representation of the results of toxic
siRNA having 19 base pairs and corresponding siRNA having 17 base
pairs. As the graph illustrates, the siRNA having 17 base pairs
resulted in significantly less toxicity. This suggests that entry
and/or processing by RISC is necessary for siRNA to induce
toxicity. Thus, preferably the toxic control siRNA is at least 18
base pairs, and more preferably at least 19 base pairs. Together
these results demonstrate that siRNA induced off-target effects can
generate measurable phenotypes.
Example 6
[0162] Three different lipids were studied on two different cell
lines to optimize an RTF protocol. Accordingly, positive control
siRNAs (e.g., cyclo3) at various concentrations were dried on well
floors of 96-well plates such that final concentrations varied
between 0 and 250 nM. Lipid solutions of OLIGOFECTAMINE.TM.,
DharmaFECT.TM. 1, or TBio were mixed with Opti-MEM.TM. and tested
on 10,000 A549 cells, or 5,000 3T3L1 cells.
[0163] FIG. 9A is a graphical representation of the toxicity of the
lipid in the A549 cells. OLIGOFECTAMINE.TM. and TBio induced
minimal toxicity at all concentrations. DharmaFECT.TM. 1 produced
minimal toxicity at 0.125 ug of lipid per 100 uL. FIG. 9B is a
graphical representation of the gene silencing efficacy achieved
with the lipids. OLIGOFECTAMINE.TM. was shown to have inefficient
gene silencing so as to be not suitable for use in siRNA RTF of
A549 cells. DharmaFECT.TM. 1 and TBio provided excellent gene
silencing at all amounts of siRNA, and were suitable for use in
A549 cells.
[0164] FIG. 10A is a graphical representation of the toxicity of
the lipid in the cells. OLIGOFECTAMINE.TM. and TBio induced minimal
toxicity at all concentrations. DharmaFECT.TM. 1 produced minimal
toxicity at 0.125 ug of lipid per 100 uL. FIG. 10B is a graphical
representation of the gene silencing efficacy achieved with the
lipids. OLIGOFECTAMINE.TM. and Tbio each showed inefficient gene
silencing so as to be not suitable for use in siRNA RTF of 3T3L1
cells. DharmaFECT.TM. 1 provided excellent gene silencing at all
amounts of siRNA, and were suitable for use in 3T3L1 cells.
Example 7
[0165] The optimization of reagents that induce the least amount of
cell toxicity and death were studied in a RTF test protocol with
three separate lipid-media or lipid-buffer mixtures. Control siRNA
(e.g., cyclo3) at various concentrations were used with lipid
solutions of DharmaFECT.TM. 1-Opti-MEM.TM., DharmaFECT.TM.
1-HyQ-MEM.TM., or DharmaFECT.TM. 1-Hanks Balanced Salt Solution
("HBSS").
[0166] FIG. 11A is a graphical representation of the gene silencing
obtained with the foregoing lipid solutions. The gene silencing in
each culture was shown to be very similar (e.g., >80% silencing.
FIG. 11B is a graphical representation of the toxicity obtained
with the foregoing lipid solutions. The toxicity varied depending
on the lipid and media conditions. At 0.125 ug of lipid per 100 uL,
both HyQ-MEM.TM. and HBSS performed more consistently than
Opti-MEM.TM. with nearly 100% cell viability for HyQ-MEM.TM. and
HBSS compared to about 80% viability for Opti-MEM.TM.. At higher
lipid concentrations of 0.25 ug per 100 uL the differences in the
performance of Opti-MEM.TM., HyQ-MEM.TM. and HBSS are more
significant. At 0.25 ug of lipid per 100 uL, cell viability with
DharmaFECT.TM. 1-Opti-MEM.TM. solutions was approximately 60%, 75%,
50%, 50%, and 25% for plates that had been aged 1, 2, 3, 4, and 8
weeks, respectively. In contrast, cell viability of cultures
treated with DharmaFEC.TM. 1-HyQ-MEM.TM. and DharmaFECT.TM. 1-HBSS
were greater than 90%. These results identify HyQ-MEM.TM. and HBSS
as preferred reagents for siRNA RTF protocols due to greater
consistency across lipid concentrations.
Example 8
[0167] In one example, a RTF testing plate or series of plates can
be designed in order to optimize RTF with control siRNA.
Accordingly, the plates can be configured to include any of the
following variables: (1) the concentration of individual or pools
of control siRNA can be between 0.01-250 nM, more preferably
between 0.05 and 100 nM, even more preferably between 0.1 and 50
nM, still even more preferably between 0.5 and 25 nM, and most
preferably between 0.75 and 10 nM or about 1 nM; (3) the types of
polynucleotide carrier can be a lipid such as DharmaFECT.TM. 1,
DharmaFECT.TM. 2, DharmaFECT.TM. 3, or DharmaFECT.TM. 4; (3) the
concentration of the lipid polynucleotide carrier can be at
concentrations of 0.05-1 ug per 100 uL of solution, more preferably
at concentrations of 0.05-0.5 ug of lipid per 100 uL of solution,
even more preferably still at concentrations of 0.05-0.25 ug of
lipid per 100 uL of solution, and most preferably at concentrations
of 0.05-0.1 ug per 100 uL of solution; (4) the types of media
and/or buffer used to complex the lipid being preferably
Opti-MEM.TM., more preferably HyQ-MEM.TM., and most preferably
buffered salt solutions such as Hanks Buffered salt solution or
equivalent mixtures; and (5) the types and amounts of cells having
densities of 1,000 to 35,000 cells per 0.35 cm.sup.2 preferred
densities of 2,000-30,000 cells per 0.35 cm.sup.2, more preferably
2,000-20,000 cells per 0.35 cm.sup.2, even more preferably
2,000-15,000 cells per 0.35 cm.sup.2, and most preferably cell
densities of 2,000-10,000 cells per 0.35 cm.sup.2.
[0168] The present invention may be embodied in other specific
forms without departing from its spirit or essential
characteristics. The described embodiments are to be considered in
all respects only as illustrative and not restrictive. The scope of
the invention is, therefore, indicated by the appended claims
rather than by the foregoing description. All changes which come
within the meaning and range of equivalency of the claims are to be
embraced within their scope.
* * * * *