U.S. patent application number 10/404988 was filed with the patent office on 2003-08-21 for kit for direct rt-pcr on oligonucleotide-immobilized pcr microplates.
Invention is credited to Mitsuhashi, Masato.
Application Number | 20030157550 10/404988 |
Document ID | / |
Family ID | 27670367 |
Filed Date | 2003-08-21 |
United States Patent
Application |
20030157550 |
Kind Code |
A1 |
Mitsuhashi, Masato |
August 21, 2003 |
Kit for direct RT-PCR on oligonucleotide-immobilized PCR
microplates
Abstract
The entire process of reverse transcription-polymerase chain
reaction (RT-PCR) is simplified by using
oligonucleotide-immobilized microplates made of, e.g.,
polypropylene, to which oligonucleotides are securely immobilized
and which can be subjected to thermal cycles of PCR. RT-PCR is
preferably conducted in solid-phase. Capturing of mRNA and RT-PCR
can be conducted in the same plates. The cDNA synthesized from the
mRNA captured on the microplates can be used more than once.
Further, in combination with the microplates, a filter plate is
used for the preparation of cell lysates wherein target cells are
placed on the filter plate, and a lysis buffer is passed through
the cell layer on the filter to transfer cell lysate directly to
the microplate via well-to-well communication.
Inventors: |
Mitsuhashi, Masato; (Irvine,
CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
27670367 |
Appl. No.: |
10/404988 |
Filed: |
March 31, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10404988 |
Mar 31, 2003 |
|
|
|
10048800 |
Jan 31, 2002 |
|
|
|
10048800 |
Jan 31, 2002 |
|
|
|
PCT/US98/27293 |
Dec 22, 1998 |
|
|
|
60068394 |
Dec 22, 1997 |
|
|
|
60071627 |
Jan 16, 1998 |
|
|
|
Current U.S.
Class: |
435/6.12 ;
435/287.2 |
Current CPC
Class: |
C12Q 2565/501 20130101;
C12Q 2521/107 20130101; C12Q 1/6806 20130101; C12N 1/06 20130101;
C12Q 1/6837 20130101; C12Q 1/686 20130101; C12Q 1/686 20130101 |
Class at
Publication: |
435/6 ;
435/287.2 |
International
Class: |
C12Q 001/68; C12M
001/34 |
Claims
What is claimed is:
1. A kit for direct RT-PCR, comprising: an
oligonucleotide-immobilized microplate for capturing mRNA from a
cell lysate, said microplate having wells to which oligonucleotides
are securely immobilized, said microplate having heat-stability for
thermal cycles of PCR, said oligonucleotides having nucleic acid
sequences specifically complimentary to mRNA of interest present in
the cell lysate; and a filter plate for preparing a cell lysate,
said filter plate having wells with a membrane having a pore size
such that target cells are trapped, but cytosolic mRNA present in
the cells passes therethrough, wherein the target cells are placed
on the membrane, said filter plate having a configuration such that
the filter plate can be mounted on top of the microplate to
establish well-to-well communication between the filter plate and
the microplate; wherein the filter plate is mounted on top of the
microplate when in use, and an appropriate lysis buffer is passed
through the cell layer on the membrane without disturbing the cells
to transfer the cell lysate to the microplate, followed by RT-PCR
using the microplate.
2. A kit for direct RT-PCR according to claim 1, wherein the
microplate is made of polypropylene or polyolefine.
3. A kit for direct RT-PCR according to claim 1, wherein the filter
plate is made of glass fiber.
4. A kit for direct RT-PCR according to claim 1, further comprising
a lysis buffer for releasing cytosolic mRNA present in the cells
when passing through the cell layer on the filter plate, said lysis
buffer comprising a mild detergent for destructing cell membranes
but maintaining nuclei to be intact and a reagent for inhibiting
Rnase activity or inactivating Rnase, said lysis buffer having a pH
and salt concentration for hybridization.
Description
BACKGROUND OF THE INVENTION
[0001] Polymerase chain reaction (PCR) following cDNA synthesis
from mRNA (reverse transcription-polymerase chain reaction, RT-PCR)
to analyze gene expression of any specific mRNA in cells and
tissues has become common technique, because of its better
sensitivity and less labor-intensive manipulations than the
traditional Northern blot (Kawasaki E S, Wang A M, "Detection of
gene expression. In: Erlich, EA. PCR technology", New York:
Stockton, 1989;89-97). Furthermore, because recently available
recombinant Tth thermostable polymerase has activities of both
reverse transcriptase and DNA polymerase, both steps can be
performed simultaneously in a single tube without changing the
buffer system (Myers T W, Gelfand D H, "Reverse transcription and
DNA amplification by a Thermus thermophilus DNA polymerase",
Biochemistry 1991;30:7661-6). However, it still requires
purification of total RNA or mRNA from cells and tissues, which
takes additional time-consuming, and labor-intensive step(s).
[0002] It has been reported that mRNA is successfully captured by
an oligo(dT)immobilized polystyrene (PS) microplate
(GENEPLATE.RTM., Hitachi Chemical Co., Japan, and AGCT, Irvine,
Calif.) (Mitsuhashi M, et al., "Gene manipulation on plastic
plates", Nature 1992:357:519-20, Miura Y, et al., "Fluorometric
determination of total mRNA with oligo(dT) immobilized on
microtiter plates", Clin Chem 1996:42:1758-64, Miura Y, et al.,
"Rapid cytocidal and cytostatic chemosensitivity test by measuring
total amount of mRNA", Cancer Lett 1997:116:139-44) followed by
single- and double-stranded cDNA synthesis on the plate (Tominaga
K, et al., "Colorimetric ELISA measurement of specific mRNA on
immobilized-oligonucleotide-coated microtiter plates by reverse
transcription with biotinylated mononucleotides", Clin Chem
1996:42:1750-7). Once double stranded cDNA is formed on a PS
microplate of GENEPLATE.RTM., sense stranded cDNA can be removed
and used as a template for multiple PCR experiments (Ishikawa T, et
al., "Construction of cDNA bank from biopsy specimens for multiple
gene analysis of cancer", Clin Chem 1997:43:764-70). Unfortunately,
PCR cannot be performed on this PS microplate, because of its heat
instability during the denaturing step in PCR cycles. Although heat
stable polypropylene (PP) tubes and microplates are primary vessels
for PCR, it is difficult to immobilize oligonucleotides onto a PP
surface, because of its extremely chamically stable surface
characteristics. However, oligo(dT)-immobilized polypropylene
plates have recently become available.
[0003] As described above, RT-PCR is a very useful technology in
various fields including diagnostic molecular pathology (Bortolin
S, et al., "Quantitative RT-PCR combined with time-resolved
fluorometry for determination of BCR-ABL mRNA", Clin Chem
1996:42:1924-9). However, there are many steps involved in the
analysis of RT-PCR; collection of cells, purification of RNA/mRNA,
cDNA synthesis, PCR, and quantitation of PCR products. In
particular, the purification of intact RNA molecules is the
critical first step for the successful RT-PCR, and this requires
labor-intensive multiple manipulations to eliminate or inactivate
endogenous or contaminated ribonuclease (RNase) in cells and
tissues. Although recent PCR technologies allow researchers to
continuously monitor the quantity of PCR products with various
in-line or off-line verification procedures of amplified PCR
products (Morris T, et al., "Rapid reverse transcription-PCR
detection of hepatitis C virus RNA In serum by using the TaqMan
fluorogenic detection system", J Clin Microbiol 1996:34:p2933-6,
Wittwer C T, et al., "The LightCycler: A microvolume multisample
fluorimeter with rapid temperature control", BioTechniques
1997:22:171-181), lack of a simplified RNA preparation system
prevents full automation of RT-PCR.
SUMMARY OF THE INVENTION
[0004] In order to simplify the entire process of gene expression
analysis, by using oligonucleotide-immobilized microplates to which
oligonucleotides are securely immobilized and which can be
subjected to thermal cycles of PCR (PCR microplates), capturing of
mRNA and reverse transcription-polymerase chain reaction (RT-PCR)
can be conducted on the same plates. Heretofore, microplates were
never used for PCR due to insufficient thermal stability, and thus,
RT-PCR processes were time-consuming and labor-intensive. In using
PCR microplates such as those made of polypropylene, polyolefine,
or polycarbonate, because of their fluorescent characteristics,
immobilized oligonucleotide, hybridized mRNA, and synthesized cDNA
are quantitated fluorometrically by using nucleic acid stain or
enzymatically by producing fluorescence or chemiluminescence. The
PCR microplates can also capture mRNA from crude cell lysates
without purification of RNA or mRNA.
[0005] Although hybridized mRNA can be successfully used for
one-step RT-PCR with rTth polymerase or two-step RT-PCR with
reverse transcription followed by PCR, two-step RT-PCR exhibits
surprisingly higher sensitivity than one-step RT-PCR. This is
surprising because two-step RT-PCR requires an inefficient solid
phase reverse transcription reaction, whereas one-step RT-PCR is
conducted in a more efficient liquid phase reaction by first
dissociating mRNA from the PCR microplates.
[0006] In addition, cDNA synthesized from mRNA captured by
immobilized oligonucleotide on the PCR microplates can be used more
than once, thereby amplifying plural times different or the same
portions of the cDNA by using appropriate primers. This
multiple-PCR system, wherein multiple PCRs are synthesized from the
cDNA on the PCR microplates, is useful in basic research,
diagnostics, and drug screening, with potential application to
future automation.
[0007] Further, conventionally, cell lysate is prepared by vigorous
homogenization processes which are not only time-consuming and
labor-intensive, but also cause fluctuation of the amount of
recovered mRNA. In the present invention, in combination with the
PCR microplates, by placing target cells on a filter plane evenly
and passing a lysis buffer through the cell layer on the filter
without disturbing the cells, it is possible to drastically
simplify the preparation of cell lysate and significantly stabilize
the yield of recovered cytosolic RNA. It is very convenient if the
microplate and the filter plate are available as a kit for direct
RT-PCR. In the above, a lysis buffer, wash buffer, reagents for
RT-PCR/PCR, and PBS are commercially available or can readily be
prepared so that they need not be included in the kit. However, for
convenience, a lysis buffer may be included in the kit for
releasing cytosolic mRNA present in the cells when passing through
the cell layer on the filter plate. In the above, the lysis buffer
comprises a mild detergent for destructing cell membranes but
maintaining nuclei to be intact and a reagent for inhibiting RNase
activity or inactivating RNase, said lysis buffer having a pH and
salt concentration for hybridization.
[0008] Because of the above features, the PCR microplates and
filter plates can drastically and surprisingly simplify the entire
process of RT-PCR from the preparation of cell lysate to measuring
specific PCR products with high reliability. Thus, this technology
is highly useful in various molecular analyses including basic
research, diagnostics, and drug screening, with potential
application to future automation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 is a graph showing quantities of oligo(dT)
immobilized onto oligonucleotide-immobilized
polystyrene/polyolefine microplates, indicating that approximately
69% of applied oligonucleotides (10 pmol) were immobilized onto the
surface of the microplates.
[0010] FIG. 2A is a graph showing mRNA specificity of the PCR
microplates, wherein YOYO-1 fluorescent intensity indicates high
specificity to mRNA (.circle-solid.) over DNA (.box-solid.), rRNA
(.DELTA.), and tRNA (.gradient.).
[0011] FIG. 2B is a graph showing mRNA specificity of the PCR
microplates, wherein alkaline phosphatase substrate (ATTOPHOS.TM.)
indicates high specificity to mRNA (.circle-solid.) over DNA
(.box-solid.), rRNA (.DELTA.), and tRNA (.gradient.).
[0012] FIG. 3 is a graph showing reversible hybridization of mRNA
on the PCR microplates, wherein YOYO-1 fluorescent intensity
indicates sufficient reversibility of mRNA hybridization.
[0013] FIG. 4A shows the results of agarose gel electrophoresis
showing RT-PCR products from the captured mRNA in crude cell
lysates with the expected size of 168 base pairs.
[0014] FIG. 4B shows the results of agarose gel electrophoresis
showing insignificant false PCR from contaminated genomic DNA in
the plates, wherein the band disappeared after being washed with
boiling DEPC (diethylpyrocarbonate) water, but not after being
washed with 55.degree. C. DEPC water.
[0015] FIG. 4C shows the results of agarose gel electrophoresis
showing PCR conducted with or without reverse transcription.
[0016] FIG. 5A is a graph showing hybridized mRNA measured by
YOYO-1 at different dilutions of cell suspension. The upper inset
shows the results of agarose gel electrophoresis showing hybridized
mRNA measured by one-step RT-PCR using rTth polymerase. The lower
inset shows the results of agarose gel electrophoresis showing
hybridized mRNA measured by two-step RT-PCR using rTth
polymerase.
[0017] FIG. 5B is a graph showing hybridized mRNA measured by
ATTOPHOS.TM. fluorescence at different dilutions of cell
suspension.
[0018] FIG. 6A is a graph showing well-to-well variation of the
amounts of immobilized oligonucleotides and hybridized rabbit
globin mRNA, measured by YOYO-1 fluorescence.
[0019] FIG. 6B is a graph showing well-to-well variation of the
amounts of synthesized cDNA from captured rabbit globin mRNA
measured by ATTOPHOS.TM. fluorescence.
[0020] FIG. 7 is a graph showing well-to-well variation of the
amounts of PCR products inter-assays and intra-assays (upper and
lower insets).
[0021] FIG. 8 is a graph showing storage stability of the PCR
microplates, wherein the amounts of cDNA synthesis were determined
by ATTOPHOS.TM. fluorescence.
[0022] FIG. 9 is a graph showing measurement of mRNA and the
results of agarose gel electrophoresis of PCR amplification of
.beta.-actin from various cultured cells which were subjected to
lysis on a glass fiber filter to capture mRNA on
oligo(dT)-immobilized polypropylene microplates.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0023] PCR Microplates
[0024] In the present invention, oligonucleotide-immobilized
microplates to which oligonucleotides are securely immobilized and
which can be subjected to thermal cycles of PCR (PCR microplates)
are used, wherein the capture of mRNA and reverse
transcription-polymerase chain reaction (RT-PCR) can be conducted
on the same plates. The PCR microplates can also capture mRNA from
crude cell lysates without purification of RNA or mRNA. What makes
PCR using microplates possible is the use of PCR microplates to
which oligonucleotides are securely immobilized and which can be
subjected to thermal cycles of PCR. For example, conventional
oligonucleotide-immobilized polystyrene microplates such as
GENEPLATE.RTM. (AGCT, Irvine, Calif.) cannot be used for PCR
because polystyrene's heat stability is low. PCR microplates usable
in the present invention include, but are not limited to,
oligonucleotide-immobilized microplates made of polypropylene,
polyolefine, or polycarbonate, and other microplates made of heat
resistant polymer or resin which can be used in thermal cycles of
PCR. In the above, polyolefine microplates may be preferred due to
their surface characteristics allowing secure immobilization of
oligonucleotides. Oligonuleotides immobilized on microplates
include, but are not limited to, oligo(dT) and other
oligonuleotides specific to mRNA or target RNA. Appropriate
sequences can be identified by using HYBsimulator.TM. software
(AGCT, Irvine, Calif.) using hybridization simulation against
GenBank primate database (Mitsuhashi M, et al., "Oligonucleotide
probe design--a new approach", Nature 1994:367:759-61, Hyndman D,
et al., "Software to determine optimal oligonucleotide sequences
based on hybridization simulation data", BioTechniques
1996:20:1090-7). See also U.S. Pat. No. 5,556,749, issued Sep. 17,
1996 to Mitsuhashi M, et al., entitled "Oigo probe designstation: a
computerized method for designing optimal DNA probes", which is
incorporated hereby by reference herein. The amount of immobilized
oligonucleotides can be as high as 10-100 pmol per well (normally
10-30 pmol).
[0025] Because of stable surface characteristics of polypropylene,
oligonucleotides cannot be immobilized thereon easily. However,
recently, some manufacturers produce PCR microplates for molecular
biological applications, which can allow researchers to conduct
high throughput PCR. In addition, a company such as Hitachi
Chemical Research Center (Irvine, Calif.) can pretreat any
commercially available PCR plates to allow oligonucleotides to be
immobilized thereonto. Accordingly, this
oligonucleotide-immobilized polypropylene (or polyolefine or
polycarbonate) plate has recently become available (AGCT, Irvine,
Calif.).
[0026] As compared with PS plates or tubes unsuitable for
94.degree. C. heat denaturing step in PCR, PCR microplates can
advantageously be used for PCR. Like widely used PCR microtubes in
molecular biological experiments, PCR microplates have low capacity
for nonspecific absorption of proteins and DNA/RNA, and resistance
to organic chemicals (i.e. phenol/chloroform). These
characteristics can be maintained even when oligonucleodites are
immobilized thereonto.
[0027] Another advantage of oligonucleotide-immobilized PCR
microplates is the strict specificity to mRNA, but not to rRNA,
tRNA or DNA, eliminating the potential problem of false PCR
amplification from contaminated genomic DNA, whereas cellulose or
beads often contain detectable amounts of rRNA, tRNA and DNA.
Furthermore, less variation among wells and plates, excellent
stability, and availability of various quality control protocols
make this technology very competitive.
[0028] Oligonucleotide-immobilized polystyrene microplates have
been, shown to exhibit a wide variety of applications, which
include the capture of total and specific mRNA, ss-cDNA and ds-cDNA
synthesis, quantitation of specific mRNA, and sense and antisense
mRNA synthesis. oligonucleotide-immobilized PCR microplates can
also be used for the same purposes as PS microplates. See U.S. Pat.
No. 5,656,462, issued Aug. 12, 1997 to Keller C, et al., entitled
"Method for synthesizing polynucleotide immobilized support", which
is incorporated hereby by reference herein.
[0029] An interesting feature of PCR microplates is their
fluorescent characteristics. Although PCR plates are cloudy and not
completely transparent compared to PS plates, fluorescence
measurement of YOYO.TM.-1 or equivalent dyes exhibited better
performance in PCR microplates than in PS microplates (Ogura M,
Mitsuhashi M, "Screening method for a large quantity of polymerase
chain reaction products by measuring YOYO-1 fluorescence on 96-well
polypropylene plates", Anal Biochem 1994:218:458-9). See also U.S.
Pat. No. 5,545,528, issued Aug. 13, 1996 to Mitsuhashi M, et al.,
entitled "Rapid screening method of gene amplification products in
polypropylene plates", which is incorporated hereby by reference
herein. This allows for conducting various analysis quite easily.
For example, the amounts of both immobilized oligonucleotides,
i.e., captured mRNA and synthesized cDNA, can be determined
fluorometrically without using radioactive materials.
[0030] Detection of Fluorescence
[0031] Because of the fluorescent characteristics of polypropylene,
polyolefine, or polycarbonate plates, immobilized oligonucleotide,
hybridized mRNA, synthesized cDNA, and PCR products are quantitated
fluorometrically by using nucleic acid stain or enzymatically by
producing fluorescence or chemiluminescence, e.g., ATTOPHOS.TM. or
LUMIPHOS.TM.. The nucleic acid stains include, but are not limited
to, a fluorescent dye selected from the group consisting of
1,1,'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3--
dihydro-(benzo-1,3-oxazole)-2-methylidene]-quinoliumetraiodide
(YOYO.TM.-1),
1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[-
3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene]-quinoliumetraiodi-
de (TOTO.TM.-1),
1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-
-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-propenylidene]-quinoliumetra-
iodide (TOTO.TM.-3), SYBR.RTM.-Green I, II, and PicoGreen.RTM.. See
also U.S. Pat. No. 5,545,528 described above. Accordingly, this
oligonucleotide-immobilized polypropylene, polyolefine, or
polycarbonate plate has recently become available.
[0032] In the following experiments, as a
oligonucleotide-immobilized PCR microplate, oligo(dT)-immobilized
polypropylene/polyolefine microplate GENEPLATE.RTM.-PP (Hitachi
Chemical Research Center, Irvine, Calif.) was used. However,
oligonucleotide-immobilized PCR microplates are not limited to the
above, and include any oligonucleotide-immobilized microplates to
which oligonucleotides are securely immobilized and which can be
subjected to thermal cycles of PCR.
[0033] Two-Step RT-PCR or Solid-Phase RT-PCR
[0034] There are roughly two ways to perform RT-PCR: one-step
RT-PCR and two-step RT-PCR. In one-step RT-PCR, with rTth
polymerase or optimal combination of reverse transcriptase and DNA
polymerase (Titan.TM., one tube RT-PCR kit, Boehringer Mannheim,
Indianapolis, Ind.), RT-PCR can be performed on the
oligonucleotide-immobilized PCR microplate without changing the
buffer. As another one-step RT-PCR, after capturing mRNA by
hybridization of mRNA and immobilized oligo(dT), mRNA is removed to
a RT-PCR buffer, and RT-PCR can be performed. In contrast, in
two-step RT-PCR, after hybridization on oligonucleotide-immobilized
PCR microplates, captured mRNA is reverse transcribed to cDNA on
the same microplates, reactants are removed by aspiration, and PCR
is conducted by using heat stable DNA polymerase, e.g., either rTth
or Taq polymerase with an appropriate buffer. In the above, PCR is
conducted in a thermal cycler with, e.g., 60 cycles of 94.degree.
C. denaturation for 1 min, 60.degree. C. annealing for 1 min
followed by 72.degree. C. extension for 1 min, by using the same
oligonucleotide-immobilized PCR microplates as used for
hybridization. It is believed that one of ordinary skill in the art
expects that one-step or liquid-phase RT-PCR is better than
two-step RT-PCR with respect to PCR efficiency. However,
surprisingly, when PCR is conducted from synthesized cDNA on the
oligonucleotide-immobilized PCR microplates (two-step RT-PCR),
RT-PCR can be approximately 10,000-fold more sensitive than
conventional one-step RT-PCR, and transcript can be detected from
cell lysates containing only 100 cells. This is very surprising
because two-step RT-PCR requires inefficient solid phase reverse
transcription reaction, whereas one-step RT-PCR is conducted in
more efficient liquid phase reaction by first dissociating mRNA
from the oligonucleotide-immobilized PCR microplates. One
explanation may be as follows: It is believed that primers are used
for dimer formation during reverse transcription. More primer
dimers are formed in one-step PCR than two-step RT-PCR. Although
hybridization efficiency is lower in two-step RT-PCR than one-step
RT-PCR, primer dimers formed during initial reverse transcription
phase exist throughout PCR in two-step RT-PCR, thereby drastically
increasing RT-PCR sensitivity.
[0035] Reamplification by Immobilized cDNA (Multiple-PCR
System)
[0036] cDNA synthesized from mRNA captured by immobilized
oligonucleotide on the oligonucleotide-immobilized PCR microplate
can be used more than once, i.e., cDNA on the
oligonucleotide-immobilized PCR microplate is quite stable. This
interesting feature allows reamplification by the same immobilized
cDNA to amplify plural times different or the same portions of the
cDNA of interest by using appropriate primers. This multiple PCR
system, wherein multiple PCRs are synthesized from the cDNA on the
oligonucleotide-immobilized PCR microplates, is useful in basic
research, diagnostics, and drug screening, with potential
application to future automation.
[0037] High Throughput RT-PCR System
[0038] Conventionally, cell lysate is prepared by disrupting cells
with a lysis buffer to release cytosolic mRNA, followed by
centrifugation. Supernatants are used for hybridization. This
vigorous homogenization process is not only time-consuming and
labor-intensive, but also causes fluctuation of the amount of
recovered mRNA. By placing cells on a filter membrane evenly and
passing a lysis buffer through the cell layer on the filter
membrane without mechanical homoginization of the cells, it is
possible to drastically simplify the preparation of cell lysate and
significantly stabilize the yield of recovered cytosolic RNA.
[0039] Namely, in the present invention, in order to simplify the
entire RT-PCR process starting from cells on culture plates, cells
are transferred to a filter plate and cells are trapped onto its
membrane by vacuum aspiration, positive pressure, or
centrifugation. The filter plate is then assembled on top of a
oligonucleotide-immobilized PCR microplate having plural wells, and
Lysis buffer is added to each well to mildly destruct cell
membranes. After these two-plate sandwiches are centrifuged, cell
lysate containing cytosolic mRNA are transferred to the
oligonucleotide-immobilized PCR microplate for hybridization. After
hybridization at room temperature for 1 hour, for example, RT-PCR
can be conducted in automated instrument. In the above, a filter or
membrane of the filter plate includes, but is not limited to, glass
fiber, polypropylene or polyolefine mesh, wool, and other membranes
which have a pore size such that target cells can be trapped
without any leakage of cells from the membrane, but cytosolic mRNA
can pass therethrough. For example, using glass fiber (Grade 934AH,
Cambridge Technology, Inc. Watertown, Mass.) or Whatman GF/F grade
glass fiber memberane, most of cultured cells and blood leukocyte
can be trapped. In the above, glass fiber plates are preferable.
Further, because the filter plate is mounted on the top of a
oligonucleotide-immobilized PCR microplate, the configuration of
the filter plate needs to correspond to that of the
oligonucleotide-immobilized PCR microplate with respect to, e.g.,
the number of wells wherein the wells of the filter plate are
communicated with the respective wells of the
oligonucleotide-immobilized PCR microplate when subjected to
centrifugation. The maximum capacity of cells per well is normally
10.sup.4 to 10.sup.7.
[0040] In the above, the cell lysate can be passed through the
membrane of the filter plate with the aid of force generated by
means of centrifugation, vacuum, or positive pressure. The force
necessary to pass the cell lysate through the membrane is easily
determined by simple experiments.
[0041] In the above, the lysis buffer comprises a detergent for
destructing cell membranes, RNase inhibitor for inhibiting RNase
activity or deactivating or destroying RNase, and pH control agent
and salt for hybridization. In the above, RNase must be active in
the lysis buffer. Further, in order to mildly destruct cell
membranes so as to prevent contamination of nucleus, the use of a
mild detergent is preferable (e.g., NP-40 or TRITON.TM.-X). The
above-described lysis buffer is useful and can be used for
oligonucleotide-immobilized PCR microplates without the use of
filter plates.
[0042] The protocols of this system include, but are not limited
to, the following:
[0043] Step 1: Transfer Cells from Culture Plate to Filter
Plate
[0044] 1. Place a filter plate onto a vacuum manifold.
[0045] 2. Transfer cells from culture plates to the filter plate by
using, e.g., multi-channel pipettes.
[0046] 3. Vacuum aspirate the filter plate to trap cells onto
membranes.
[0047] 4. Wash each well once or twice with, e.g., 50 .mu.l each of
PBS (optional).
[0048] Step 2: Transfer Cell Lysate from Filter Plate to
Oligonucleotide-Immobilized PCR Microplate for Hybridization
[0049] 1. Remove the filter plate from the vacuum manifold and
place it on top of the oligonucleotide-immobilized PCR
microplate.
[0050] 2. Add, e.g., 50 .mu.l of lysis buffer (e.g., 10 mM Tris, pH
8.0, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl
ribonucleoside complex, RNase-free), and centrifuge at, e.g.,
1,500.times.g for 10 min.
[0051] 3. Incubate the oligonucleotide-immobilized PCR microplate
at room temperature for 1 hour for hybridization between
immobilized oligo(dT) and poly(A) tails of mRNA present in
cytosolic fraction of cells.
[0052] Step 3: RT-PCR and Post-PCR Analysis
[0053] 1. Wash each well once or twice with, 50 .mu.l of wash
buffer (e.g., 10 mM Tris, pH 8.0, 1 mM EDTA, 0.3 M NaCl,
RNase-free).
[0054] 2. Start RT-PCR and monitor the amount of PCR products in an
automated PCR instrument.
[0055] According to the present invention, a rapid, inexpensive,
high throughput, and easily automated method for the entire RT-PCR
process starting from cells can be realized.
[0056] Philadelphia chromosome (Ph.sup.1) found frequently in
chronic myelogenous leukemia (CML) is a reciprocal translocation of
ABL protooncogene from chromosome 9 to a portion of the BCR gene in
chromosome 22 [t(9;22)(q34;q11)] (Wehnert M S, et al., "A rapid
scanning strip for triand dinucleotide short tandem repeats",
Nucleic Acids Res 1994:22:1701-4). Specific RT-PCR amplification of
BCR-ABL mRNA from peripheral blood cells or bone marrow cells
provides a highly sensitive and quantitative methodology for the
detection of residual leukemic cells. Because the detection of
residual leukemic cells is one of critical indicators for the
treatments of CNE, RT-PCR test of BCR-ABL mRNA is widely available
in many institutions. However, in many cases, total RNA or mRNA is
first purified from cell suspension. Using the present system, once
cell lysates are applied to the oligonucleotide-immobilized PCR
microplates for hybridization, one can proceed with not only direct
RT-PCR described here, but also YOYO.TM.-1 quantitation of total
amounts of mRNA (Miura Y, et al., Clin Chem 1996:42:1758-64), which
may provide additional means of normalization or quality control of
tested materials. Because of its simplicity and fluorescent
characteristics, oligonucleotide-immobilized PCR microplates may be
accepted as a platform for various molecular analyses including
basic research, diagnostics, and drug screening, with potential
application to future automation, especially in combination with
filter plates.
EXAMPLES
[0057] The invention will be further explained with reference to
Examples shown below. Materials and methods used in the Examples
are as follows:
[0058] Materials: Oligo(dT)-immobilized oligonucleotide-immobilized
PCR microplates (GENEPLATE.RTM.-PP, Hitachi Chemical Research
Center, Irvine, Calif.), YOYO.TM.-1
(1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)--
bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-oxazole)-2-methylidene]-quinoliumet-
raiodide, Molecular Probes, Eugene, Oreg.), reagents for PCR (Taq
polymerase, EZ rTth RNA-PCR kit) (Perkin Elmar, Foster City,
Calif.), K562 cell line (American Type Culture Collection,
Rockville, Md.), 100 bp DNA ladder, phosphate buffered saline
(PBS), vanadyl ribonucleoside complex (VRC), rabbit globin mRNA,
cell culture medium and appropriate antibiotics, buffer-saturated
phenol (Gibco-BRL, Geithersburg, Md.), fetal bovine serum (FBS,
HyClone, Logan, Utah), biotin-dUTP (Clontech, Palo Alto, Calif.),
ATTOPHOS.TM. (alkaline phosphatase substrate, JBL Scientific, San
Luis Obispo, Calif.), cd4465 DNA (Genome Systems, St. Louis, Mo.)
were purchased from the designated suppliers. RNA preparation
reagents for MAGEXTRACTOR.TM. were kindly provided by Toyobo
(Osaka, Japan). All other chemicals were purchased from Sigma (St.
Louis, Mo.).
[0059] Cell culture: K562 calls were grown in RPMI 1640 containing
10% FBS, 500 units/ml penicillin, 500 .mu.g/ml streptomycin, and
subcultured twice a week at a ratio of approximately 1:10. Cell
viability was assessed by the exclusion of trypan blue, and was
always more than 95%.
[0060] Preparation of cell lysate and total RNA: Cells were washed
with PBS twice, and suspended in lysis buffer (10 mmol/L Tris, pH
7.6, 1 mmol/L EDTA, 0.1% NP-40 and 20 mmol/L VRC) on ice for 5 min
to release cytosolic mRNA as previously described (Miura Y, et al.,
Clin Chem 1996:42:1758664). Samples were then centrifuged at
15,000.times.g at 4.degree. C. for 5 min, and supernatants were
applied to GENEPLATE.RTM.-PP for hybridization. In some
experiments, cells were suspended in VRC-free lysis buffer, and
immediately treated with an equal volume of phenol/chloroform twice
to absorb proteins and nucleases. Deproteinated solutions were then
subjected to hybridization.
[0061] When a glass fiber filter plate having 96 wells, adapted to
be placed on the top of the microplates, was used to recover RNA,
the above vigorous homogenization process was entirely omitted.
Culture cells were transferred to the glass fiber filter plate by
using multi-channel pipettes (described later).
[0062] Total RNA was prepared by automated instrument
(MAGEXTRACTOR.TM. MFX-2000, Toyobo, Osaka, Japan). In brief, cell
pellets were suspended in kit-supplied caotropic agents, and placed
in MAGEXTRACTOR, where RNA was absorbed to the surface of silica
felite particles followed by magnet separation. RNA was then
automatically eluted to 40 .mu.l of low salt buffer, and was stored
at -80.degree. C. in a freezer until use. The final RNA was
analyzed by agarose gel electrophoresis to confirm 18s and 28s rRNA
bands.
[0063] Primer design and synthesis: Primers for cd4465 (sense:
5'-agtttcggagcggatgaatgc-3', antisense:
5'-ggggcatcagaattttggttga-3'), rabbit globin mRNA (sense:
5'-cgtggagaggatgttcttgg-3', antisense:
5'-aacgatatttggaggtcagcac-3') and bcr-abl (sense:
5'-gaccaactcgtgtgtgaaac- tcca-3', antisense:
5'-aaagtcagatgctactggccgct-3') were designed by HYBsimulator.TM.
software (AGCT, Irvine, Calif.) using hybridization simulation
against GenBank primate database (Mitsuhashi M, et al., Nature
1994:367:759-61, Hyndman D, et al., BioTechniques 1996:20:1090-7).
In the case of bcr-abl, the sense primer was located at bcr axon 2
and the antisense primer was located at abl axon 2. Primers were
synthesized by DNA synthesizer (380B, Applied Biosystem, Foster
City), according to the manufacturer's protocol.
[0064] One-Step RT-PCR: Template DNA/RNA, 300 .mu.mol/L each of
dATP, dGTP, dCTP and dTTP, 1.times. EZ buffer, 2 mmol/L
Mn(OAc).sub.2, 0.5 .mu.mol/L each of primer, and 0.1 .mu.l rTth
polymerase were mixed in a final volume of 5-50 .mu.l, and
overlayered with one drop of nuclease free mineral oil (Sigma). PCR
was conducted in a thermal cycler (MJ Research, Watertown, Mass.)
with 1 cycle of reverse transcription at 60.degree. C. for 30 min
and 94.degree. C. denaturation for 1 min, followed by 40 cycles of
60.degree. C. annealing/extension for 1 min and 94.degree. C. of
denaturation for 1 min. After PCR was completed, PCR products were
analyzed by a 2.0% agarose gel electrophoresis with 0.5 .mu.g/ml
ethidium bromide in an electrophoresis chamber. Photographic images
were recorded on Polaroid films (667, Cambridge, Mass.).
[0065] Two-Step RT-PCR: After hybridization, captured mRNA was
reverse transcribed to cDNA by replacing biotin-dUTP with 10 mmol/L
dTTP. Reactants were removed by aspiration, and PCR was conducted
by using either rTth or Taq polymerase. For PCR with Taq
polymerase, the buffer contained 1.times. buffer, 1.25 mmol/L
MgCl.sub.2, 300 .mu.mol/L each of dATP, dGTP, dCTP and dTTP, 0.5
.mu.mol/L each of primer, and 0.5 .mu.l Taq polymerase in a final
volume of 10-50 .mu.l. PCR was conducted in a thermal cycler (MJ
Research) with 60 cycles of 94.degree. C. denaturation for 1 min,
60.degree. C. annealing for 1 min followed by 72.degree. C.
extension for 1 min.
[0066] Experiment 1: Quantitation of Immobilized
Oligonucleotides
[0067] GENUNC.TM. PP microplates (Nunc, Naparville, Ill.) treated
at Hitachi Chemical Research Center, Irvine, Calif., were obtained
from AGCT, Irvine, Calif., and oligonucleotides were immobilized
thereonto. Oligonucleotide concentrations were determined before
and after immobilization as 1.0 OD.sub.260 unit equals to 30
.mu.g/ml, and the amounts of immobilized oligonucleotides were
calculated by subtracting one value from another. In separate
experiments, YOYO-1 was diluted in TE (10 mmol/L Tris, pH 8.0, 1
mmol/L EDTA) in a final dilution of 1:1000, and applied to
GENEPLATE.RTM.-PP microplates. The fluorescence was determined by
CYTOFLUOR.TM. 2300 (Millipore, Bedford, Mass.) with excitation and
emission wavelengths of 485 nm (bandwidth 20 nm) and 530 nm (band
width 25 nm), respectively, as previously described (Miura Y, et
al., Clin Chem 1996:42:1758-64, Miura Y, at al., Cancer Lett
1997:116:139-44).
[0068] FIG. 1 is a graph showing quantities of oligo(dT)
immobilized onto the oligonucleotide-immobilized PCR microplates.
Oligonucleotide concentrations were determined before and after
immobilization as 1.0 OD.sub.260, and the amounts of immobilized
oligonucleotides were calculated by subtracting one value from
another (.largecircle.). In parallel experiments, 1:1000 dilution
of YOYO-1 was added to each well and its fluorescence was
determined (.circle-solid.). Bars indicate the % immobilization
from applied oligo(dT).sub.20. Each data point was the mean from
triplicate determinations. Although the oligonucleotide-immobil-
ized PCR microplates were opaque and not transparent compared to
conventional PS plates, as shown in FIG. 1 (.circle-solid.), the
fluorescence of the oligonucleotide-immobilized PCR microplates was
significantly increased and reached a plateau when more than 10
pmol of oligonucleotides were applied. In further quantitating the
actual amounts of immobilized oligonucleotides on the
oligonucleotide-immobilized PCR microplates, as shown in FIG. 1
(.largecircle.), 21.1 pmol of oligonucleotides were immobilized
after 100 pmol of oligonucleotides were applied to each well. The
amounts of immobilized oligonucleotides were saturated to 10-20
pmol, when more than 10 pmol of oligonucleotides were applied.
Approximately 69% of applied oligonucleotides (10 pmol) were
immobilized onto the surface of the microplates (FIG. 1, bar
graph).
[0069] Experiment 2: mRNA Specificity of
Oligonucleotide-Immobilized PCR Microplates
[0070] The next series of experiments was conducted to show mRNA
specificity. FIG. 2A is a graph showing mRNA specificity of the
oligonucleotide-immobilized PCR microplates, wherein YOYO-1
fluorescent intensity indicates high specificity to mRNA
(.circle-solid.) over DNA (.box-solid.), rRNA (.DELTA.), and tRNA
(.gradient.). FIG. 2B is a graph showing mRNA specificity of the
oligonucleotide-immobilized PCR microplates, wherein substrate
ATTOPHOS.TM. indicates high specificity to mRNA (.circle-solid.)
over DNA (.box-solid.), rRNA (.DELTA.), and tRNA (.gradient.).
[0071] In the figures, various concentrations of rabbit globin mRNA
(.circle-solid.), DNA (.box-solid.), rRNA (.DELTA.), tRNA
(.gradient.) were suspended in 50 .mu.l of hybridization buffer (10
mmol/L Tris, pH 8.0, 1 mmol/L EDTA, 0.5 M NaCl) and applied to the
well of the oligonucleotide-immobilized PCR microplates. After
hybridization at room temperature for 1 hour, each well was washed
once with hybridization buffer. In FIG. 2A, YOYO.TM.-1 was diluted
in TE (10 mmol/L Tris, pH 8.0, 1 mmol/L EDTA) in a final dilution
of 1:1000, and applied to each well, and the fluorescence was
determined by CYTOFLUOR.TM. 2300. In FIG. 2B, each well was
resuspended in 50 .mu.l of cDNA synthesis buffer (50 mmol/L Tris,
pH 8.3, containing 75 mmol/L KCl, 3 mmol/L MgC12, 10 mmol/L DTT, 10
mmol/L each of dATP, dGTP, dCTP, 250 .mu.mol/L biotin-dUTP, and 400
U of MMLV reverse transcriptase), and incubated at 37.degree. C.
for 1 hour. After each well was washed three times with wash buffer
(10 mmol/L Tris, pH 7.6, containing 300 mmol/L NaCl and 10 mmol/L
Tween 20), 50 .mu.l of wash buffer containing 1:1000 dilution of
streptavidin-alkaline phosphatase conjugates and incubated at room
temperature for 30 min. After each well was washed three times with
wash buffer, 50 .mu.l of substrate (ATTOPHOS.TM.) was added and
incubated at room temperature for 20 min. The reaction was
terminated by adding an equal volume (50 .mu.l) of 100 mmol/L EDTA,
and the fluorescence was determined by CYTOFLUOR.TM. 2300. Each
data point was the mean .+-.S.D. from triplicate
determinations.
[0072] As shown in FIG. 2A, significant YOYO.TM.-1 fluorescence was
obtained from the wells where more than 100 ng of mRNA was applied,
whereas YOYO.TM.-1 fluorescence was not increased in the wells of
rRNA, tRNA and DNA even when as many as 10 .mu.g was applied. The
specificity of quantitative cDNA synthesis on the
oligonucleotide-immobilized PCR microplates was also tested as
described above. As shown in FIG. 2B, significant ATTOPHOS.TM.
fluorescence was obtained from the well where more than 0.1 ng/well
of mRNA was applied, but not from the wells of rRNA, tRNA and DNA
even when as many as 10 .mu.g was applied.
[0073] Experiment 3: Quantitation of Hybridization
[0074] FIG. 3 is a graph showing reversible hybridization of mRNA
on the oligonucleotide-immobilized PCR microplates. One .mu.g of
rabbit globin mRNA or 20 .mu.g of total liver RNA was suspended in
50 .mu.l of hybridization buffer and applied to the well of the
oligonucleotide-immobilized PCR microplates. After hybridization at
room temperature for 1 hour, wells were washed three times with
DEPC water at different temperatures (25.degree. C., 55.degree. C.
or boiling). YOYO.TM.-1 was then applied to each well, and the
fluorescence was determined by CYTOFLUOR.TM. 2300 as described
above (Miura Y, et al., Clin Chem 1996:42:1758-64). Each data point
was the mean .+-.S.D. from triplicate determinations. As shown in
FIG. 3, YOYO.TM.-1 fluorescence was reduced to the basal levels by
adding boiling DEPC water.
[0075] Experiment 4: Capacity of Hybridization
[0076] Moreover, in order to assess the hybridization capacity,
various amounts of globin mRNA, total liver RNA or cell lysates
were applied to the oligonucleotide-immobilized PCR microplates for
hybridization. Hybridized mRNA was then recovered from the plates
by adding boiling water, and a buffer (concentration was adjusted)
was applied to fresh oligonucleotide-immobilized PCR microplates
for the second hybridization. In parallel experiments, known
concentrations of globin mRNA were also applied as a control. The
quantitative cDNA synthesis described below was then conducted, and
the amount of mRNA in the solutions was determined based on the
values of standard globin mRNA. The amounts of cDNA synthesis were
quantitated according to the protocol published by Tominaga et al.
(Clin Chem 1996:42:1750-7) with minor modification. In brief, the
mRNA-hybridized oligonucleotide-immobilized PCR microplate was
resuspended in 50 .mu.l of cDNA synthesis buffer (50 mmol/L Tris,
pH 8.3, containing 75 mmol/L KCl, 3 mmol/L MgCl.sub.2, 10 mmol/L
DTT, 10 mmol/L each of dATP, dGTP, dCTP, 250 .mu.mol/L biotin-dUTP,
and 400 U of MMLV reverse transcriptase), and incubated at
37.degree. C. for 1 hour. After each well was washed three times
with wash buffer (10 mmol/L Tris, pH 7.6, containing 300 mmol/L
NaCl and 10 mmol/L Tween 20), 50 .mu.l of wash buffer containing
1:1000 dilution of streptavidin-alkaline phosphatase conjugates was
added and incubated at room temperature for 30 min. After each well
was washed three times with wash buffer, 50 .mu.l of substrate
(ATTOPHOS.TM., 1.times. concentration) was added and incubated at
room temperature for 20 min. The reaction was terminated by adding
an equal volume (50 .mu.l) of 100 mmol/L EDTA, and fluorescence was
determined by CYTOFLUOR.TM. 2300 (Millipore) with excitation and
emission wavelengths of 485 nm (bandwidth 20 nm) and 560 nm (band
width 25 nm), respectively.
[0077] As shown in Table I, approximately 50-65% of applied globin
mRNA was hybridized to the plates. Applied globin mRNA did not
saturate the plates even when 500 ng was used; 500 ng of globin
mRNA equals approximately 1-2 pmol compared to 21 pmol of
immobilized oligonucleotides. Moreover, approximately 34-48% of
total RNA or cell lysates were captured by the plates when mRNA
concentration was low, whereas high concentrations decreased
capture efficiency, probably because of inefficient hybridization
due to high viscosity.
1 TABLE I Amounts Captured mRNA of total (means .+-. S.D., Applied
mRNA n = 3) % Capture globin mRNA 500 ng 500 ng 326.7 .+-. 47.3 ng
65.3% 50 ng 50 ng 32.0 .+-. 5.6 ng 64% 5 ng 5 ng 2.5 .+-. 0.3 ng
50% liver RNA 50 .mu.g 583 ng 45.6 .+-. 14.2 ng 7.8% 5 .mu.g 58.3
ng 14.8 .+-. 5.2 ng 25.4% 0.5 .mu.g 5.83 ng 2.8 .+-. 0.3 ng 48.0%
K562 cells 10.sub.5 cells 24.3 ng 4.2 .+-. 0.9 ng 17.2% 10.sub.4
cells 2.43 ng 0.83 .+-. 0.3 ng 34.1%
[0078] As shown above, the plates are not saturated even when as
much as 500 ng of mRNA is applied, which also represents
approximately 500 .mu.g of total RNA or 10.sup.7 cells per small
surface area of 96 well plates. This is more than enough for the
majority of experiments.
[0079] Experiment 5: RT-PCR in Oligonucleotide-Immobilized PCR
Microplates
[0080] Human K562 leukemic cells, which express the b3a2 transcript
from the Ph.sup.1 translocation, were lysed with lysis buffer
followed by centrifugation to remove cell debris and nuclear DNA.
The supernatant containing cytosolic mRNA was then applied to the
oligonucleotide-immobil- ized PCR microplates for hybridization.
After 1 hour of hybridization at room temperature, unbound
materials were removed by washing with hybridization buffer twice,
and RT-PCR was started in the same wells.
[0081] That is, in FIG. 4A, 10.sup.8 K562 cells were suspended in
lysis buffer (10 mmol/L Tris, pH 7.6, 1 mmol/L EDTA, 0.1% NP-40 and
20 mmol/L VRC) on ice for 5 min to release cytosolic mRNA. Samples
were then centrifuged at 15,000.times.g at 4.degree. C. for 5 min,
and supernatants were applied to the oligonucleotide-immobilized
PCR microplates for hybridization (lane 1). In lane 2, cells were
suspended in VRC-free lysis buffer, and immediately treated with an
equal volume of phenol/chloroform twice to absorb
proteins/nucleases. Deproteinated solutions were then subjected to
hybridization. In lane 3, total RNA was prepared by automated
instrument as described in the Methods. After hybridization, RT-PCR
was conducted in a thermal cycler with 1 cycle of reverse
transcription at 60.degree. C. for 30 min and 94.degree. C.
denaturation for 1 min, followed by 40 cycles of 60.degree. C.
annealing/extension for 1 min and 94.degree. C. of denaturation for
1 min, as described in the Methods. Lane 4 was a control cd4465
DNA.
[0082] As shown in FIG. 4A (lane 1), BCR-ABL transcript was
successfully amplified from the captured mRNA in crude cell lysates
with the expected size of 168 base pairs. The size of PCR products
was identical to that of PCR products from purified total RNA in
the same cells (FIG. 4A, lane 3). Phenol/chloroform treated cell
lysates exhibited thicker PCR products than VRC-containing cell
lysates (FIG. 4A, lane 2).
[0083] In order to analyze the false PCR from contaminated genomic
DNA in the plates, mRNA was removed by 55.degree. C. or boiling
DEPC water and one-step RT-PCR was conducted. That is, in FIG. 4B,
after total RNA was hybridized, wells were washed with 55.degree.
C. DEPC water or boiling DEPC water 3 times, and one-step RT-PCR
was conducted. As shown in FIG. 4B, PCR products of BCR-ABL
transcript when washed with disappeared when wells were washed with
boiling water, but not when washed with 55.degree. C. water. These
results were comparable to that of FIG. 3.
[0084] In separate experiments, PCR was conducted with or without
reverse transcription. That is, in FIG. 4C, after total RNA was
hybridized, cDNA was synthesized in one tube (+) and one tube was
left without reverse transcription (-). PCR was then conducted with
Taq polymerase in the presence of 1.25 mM MgCl.sub.2 with 60 cycles
of 94.degree. C. denaturation for 1 min, 60.degree. C. annealing
for 1 min followed by 72.degree. C. extension for 1 were separated
by 2.0% agarose gel electrophoresis followed by staining with
ethidium bromide. Mk indicates a 100 bp ladder. As shown in FIG.
4C, PCR products of BCR-ABL transcript were not amplified from the
wells of negative reverse transcription even under low stringent
conditions with a higher Mg concentration, whereas significant
amounts of PCR products were obtained from the wells of positive
reverse transcription.
[0085] In view of the foregoing, an advantage of the
oligonucleotide-immobilized PCR microplates is the strict
specificity to mRNA, but not to rRNA, tRNA or DNA (FIGS. 2A, 2B,
4A, 4B, 4C), eliminating the potential problem of false PCR
amplification from contaminated genomic DNA, whereas cellulose or
beads often contain detectable amounts of rRNA, tRNA and DNA.
[0086] Experiment 6: Two-Step RT-PCR
[0087] Direct RT-PCR experiments were conducted at different
dilutions of cell suspension. Various numbers of K562 cells were
applied to the oligonucleotide-immobilized PCR microplates for
hybridization. The resultant hybridized mRNA was used for either
measurement of Yoyo-1, or one-step RT-PCR using rTth polymerase.
That is, in FIGS. 5A and 5B, various amounts (0-6.times.10.sup.6)
of K562 cells were suspended in lysis, and were applied to the
oligonucleotide-immobilized PCR microplates for hybridization. In
FIG. 5A, the amounts of hybridized mRNA were determined by
YOYO.TM.-1 fluorescence, as described above. In parallel
experiments, captured mRNA was immediately subjected to one-step
RT-PCR using rTth polymerase, as described above (Inset, upper). In
FIG. 5B, in another series of experiments, cDNA was synthesized
from captured mRNA using MMLV reverse transcriptase and immobilized
oligo(dT) as a primer in the presence of biotin dUTP, followed by
quantitation of cDNA synthesis, as described above. In parallel
experiments, cDNA was synthesized on the
oligonucleotide-immobilized PCR microplates by replacing
biotin-dUTP with unlabeled dTTP, and PCR was conducted with rTth
polymerase, as shown above (Inset, lower). The PCR products were
separated by 2.0% agarose gel electrophoresis followed by staining
with ethidium bromide. M indicates a 100 bp ladder. Each data point
was the mean .+-.S.D. from triplicate determinations.
[0088] As shown in FIG. 5B, significant ATTOPHOS.TM. signals were
obtained even from as few as 10.sup.4 cells, suggesting 100-fold
more sensitivity than YOYO.TM.-1. More advantageously, when PCR was
conducted from synthesized cDNA on the oligonucleotide-immobilized
PCR microplates. PCR band was detected from as few as 10 cells
(FIG. 5A, inset bottom).
[0089] In view of the foregoing, RT-PCR from synthesized cDNA on
the oligonucleotide-immobilized PCR microplates (Two-step RT-PCR,
FIG. 5A, lower inset) is approximately 100,000-fold more sensitive
than conventional one-step RT-PCR, and bcr-abl transcript was
detected from cell lysates containing only 10 cells (FIG. 5A, top
inset). This is surprising because two-step RT-PCR required
inefficient solid phase reverse transcription reaction, whereas
one-step RT-PCR was conducted in more efficient liquid phase
reaction by first dissociating mRNA from the
oligonucleotide-immobilized PCR microplates. Since rTth was used
for both experiments, the difference was not due to the enzyme.
Because more primer dimers were formed in one-step PCR than
two-step RT-PCR (FIG. 5A, top and lower insets: the clear band on
each lane in the top inset indicate primer dimers), it is believed
that primers are used for dimer formation during reverse
transcription. In two-step RT-PCR, these primer dimers can be
removed when the reaction mixture was switched from cDNA synthesis
to PCR, whereas primer dimers formed during the initial reverse
transcription phase exist throughout PCR.
[0090] Experiment 7: Intra- and Inter-Assay of Oligonucleotide
Immobilization, Hybridization, and cDNA Synthesis
[0091] In order to conduct quantitative analysis on the
oligonucleotide-immobilized PCR microplates, well-to-well variation
is a critical issue. One hundred pmol of oligonucleotides were
applied to the oligonucleotide-immobilized PCR microplates for
immobilization followed by YOYO.TM.-.sub.1 fluorescence
determination in a fluorescent plate reader, as described above
(FIG. 6A.circle-solid.). One hundred ng of rabbit globin mRNA was
applied to each well for hybridization, followed by YOYO.TM.-.sub.1
fluorescence determination in a fluorescent plate reader, as
described above (FIG. 6A.box-solid.). One hundred ng of rabbit
globin mRNA was applied to each well for hybridization, followed by
cDNA synthesis in the presence of biotin-dUTP. ATTOPHOS.TM.
fluorescence was then determined in a fluorescent plate reader, as
described above (FIG. 6B.DELTA.). Each data point was the mean
.+-.S.D. from 10 (Intra-essay) to 3 (Inter-assay) separate
determinations.
[0092] As shown in FIGS. 6A and 6B, variation of the amounts of
immobilized oligonucleotides (FIG. 6A.circle-solid.), hybridized
rabbit globin mRNA (FIG. 6A.box-solid.), and synthesized cDNA from
captured rabbit globin mRNA (FIG. 6B.DELTA.) were all less than
10-15% within a single microplate (Intra-assay) or multiple lots of
microplates (Inter-assay). More importantly, the variation of the
amount of PCR products in these intra- and inter-assays were also
within 10% (FIG. 7). In FIG. 7, one hundred ng of rabbit globin
mRNA was applied to each well for hybridization, followed by cDNA
synthesis in the presence of unlabeled dTTP. PCR was then conducted
with rabbit globin specific primers and Taq polymerase, as
described in the Methods. The PCR products were separated by 2.0%
agarose gel electrophoresis followed by staining with ethidium
bromide. Right lanes indicate a 100 bp ladder. The amounts of PCR
products were determined by measuring OD.sub.260 (.circle-solid.).
Each data point was the mean .+-.S.D. from 10 (Intra-assay) to 3
(inter-assay) separate determinations.
[0093] In view of the foregoing, less variation among wells and
plates, excellent stability, and availability of various quality
control protocols (e.g., FIGS. 6A, 6B, 7) make this technology very
competitive.
[0094] Experiment 8: Stability of Oligonucleotide-Immobilized PCR
Microplates
[0095] The oligonucleotide-immobilized PCR microplates were stored
at room temperature (.circle-solid.), 55.degree. C. (.box-solid.)
or 72.degree. C. (.DELTA.) for 2, 8 or 15 days. One hundred ng of
rabbit globin mRNA was then applied to each well for hybridization,
followed by cDNA synthesis in the presence of biotin-dUTP.
ATTOPHOS.TM. fluorescence was then determined in a fluorescent
plate reader, as described above. Each data point was the mean
.+-.S.D. from triplicate determinations.
[0096] As shown in FIG. 8, quantities of cDNA synthesis did not
show any significant decreases even after storage at 72.degree. C.
for 15 days.
[0097] Experiment 9: Multiple PCRs from cDNA Synthesized on
Oligonucleotide-Immobilized PCR Microplates
[0098] K562 cells (10.sup.4-10.sup.6) were suspended in Lysis
buffer and were applied to the oligonucleotide-immobilized PCR
microplates for hybridization. The captured mRNA was converted to
cDNA with MMLV reverse transcriptase as described above. As
controls, some wells were treated identically but without MMLV
reverse transcriptase. Then bcr-abl transcript was amplified by PCR
with Taq polymerase, as described above (1st bcr-abl). After PCR,
each well was washed with boiling DEPC water five times, and PCR
was repeated with the same primer set (2nd bcr-abl). PCR was then
repeated a third time with primer pair from G3PDH (3rd G3PDH). The
PCR products were separated by 2.0% agarose gel electrophoresis,
followed by staining with ethidium bromide. As a result, the
agarose gel electrophoresis indicates that PCR products of bcr-abl
and G3PDH transcripts were not amplified from the wells of negative
reverse transcription, indicating no "false" PCR products from
contaminating genomic cDNA in the plates. More interestingly, the
agarose gel electrophoresis confirms that bcr-abl and G3PDH
transcripts were reamplified plural times from immobilized cDNAs
from wells.
[0099] Experiment 10: Collection of Cytosolic mRNA Fraction to
Oligonucleotide-Immobilized PCR Microplates from Various Cells by
Glass Fiber Filter
[0100] Various human cultured cell lines were used in this
experiment: K562 leukemic, U937 leukemic, CaRI colon cancer, HepGII
hepatoma, KatolII stomach cancer, and CRL 5800 lung adenocarcinoma
(American Type Culture Collection, Rockville, Md.). A 96-well
filter plate with a single layer was made of glass fiber (Cambride
Technology grade 934AH, Brandel, Gaithersburg, Md.) in order to
trap cells thereon. In preliminary experiments, the maximum
capacity of cells trapped on the single layer of glass fiber filter
membranes in each well of the 96-well filter plate was determined.
Various numbers of cells (10.sup.2 to 5.times.10.sup.8) were
applied to the filter plate assembled on top of a regular 96-well
microplate, and centrifuged at 500.times.g for 10 min. The number
of cells in the passed-through fraction collected in the well of
the lower plates was measured with a hemocytometer. As a result,
the maximum capacity of cells per well was approximately
2.times.10.sup.6, 2.times.10.sup.6, 10.sup.6, 5.times.10.sup.6,
5.times.10.sup.5 and 3.times.10.sup.5 for K562, U937, CaRI, HepGII
KatolII, and CRL 5800 cells, respectively, without any leakage of
cells from glass fiber membranes.
[0101] In the next series of experiments, 10.sup.5 cells were
applied to the filter plate, and cells were trapped onto the
membrane by vacuum aspiration. The membranes were washed twice with
PBS, and placed on top of the oligo(dT)-immobilized
polypropylene/polyolefine microplate (GENEPLATE.RTM.-PP, AGCT)
which would subsequently be used as a oligonucleotide-immobilized
PCR microplate. Fifty .mu.L of lysis buffer (10 mM Tris, pH 8.0, 1
mM EDTA, 0.5 M NaCl, 0.5% NP-40 detergent, and 20 mM vanadyl
ribonucleoside complex (VRC, Gibco-BRL, Geithersburg, Md.)) was
added to each well, and was immediately centrifuged at 500.times.g
for 10 min to recover cytosolic RNA fraction into the
oligonucleotide-immobilized PCR microplate. The lysis buffer
allowed hybridization between oligo(dT) and poly(A) sequences of
mRNA in the presence of RNase inhibitor VRC. After hybridization at
room temperature for 1 hr, the oligonucleotide-immobilized PCR
microplate was washed twice with a wash buffer (10 mM Tris, pH 8.0,
1 mM EDTA, and 0.5 M NaCl). At this stage, total mRNA was captured
in each well of the oligonucleotide-immobilized PCR microplate for
analysis. Because of the heat-stable characteristics of the
oligonucleotide-immobilized PCR microplate, the
oligonucleotide-immobilized PCR microplates were directly subjected
to PCR without transfer of mRNA to PCR vessels.
[0102] Experiment 11: Measurement of mRNA and PCR Amplification of
.beta.-Actin from Various Cultured Cells
[0103] Subsequent to Experiment 10, the first analysis was to
amplify housekeeping genes from captured mRNA. The cDNA was
synthesized by adding RT-buffer (50 mM Tris, pH 8.3, containing 75
mM KCl, 3 mM MgCl.sub.2, and 10 mM DTT; 10 mM of each dNTP; and 100
U of MMLV reverse transcriptase (Gibco-BRL), and incubated at
37.degree. C. for 1 hour. PCR was then conducted on the same plate,
by replacing RT-buffer with PCR-buffer (10.times. PCR buffer, 1.5
mM MgCl.sub.2, 100 .mu.M of dNTPs, 1.5 units of Taq DNA polymerase
(Perkin Elmer, Fostercity, Calif.), 0.5 .mu.M each of upstream
sense primer and downstream antisense primer of human .beta.-actin
(Clontech, Palo Alto, Calif.) in a final volume of 20 .mu.L. PCR
was conducted in a thermal cycler (MJ Research, PTC-100, Watertown,
Mass.) with 35 cycles of 45 seconds at 94.degree. C., 45 seconds at
60.degree. C. and 2 minutes at 72.degree. C. PCR products were
analyzed in a 1.0% agarose gel electrophoresis with 0.5 .mu.g/mL
ethidium bromide. As shown in FIG. 9 (left insets: lane M, 100 bp
DNA ladder; lane 1 K562; lane 2, U937; lane 3, CaRI; lane 4,
HepGII; lane 5, KatolII; lane 6, CRL5800), .beta.-actin gene was
successfully amplified from various cultured cells. Since intron
sequences exist between sense and antisense primers, the size of
.beta.-actin PCR products were equal to that of intron-free mRNA.
Furthermore, when starting PCR without cDNA synthesis, the
.beta.-actin gene was not amplified, suggesting that PCR was
mRNA-specific, and not derived from contaminated DNA.
[0104] In parallel experiments, the amounts of captured mRNA on the
oligonucleotide-immobilized PCR microplate were quantitated by the
method published previously from our laboratory (Tominaga K, et
al., "Colorimetric ELISA measurement of specific mRNA on
immobilized-oligonucleotide-coated microtiter plates by reverse
transcription with biotinylated mononucleotides", Clin Chem
1996:1750-1757, 1996) with minor modifications. In brief, the first
strand cDNA was synthesized on the microplate by adding RT-buffer
containing 250 .mu.M biotin-dUTP instead of 10 mM of dTTP, and
incubated at 37.degree. C. for 1 hour. Each well was washed three
times with wash buffer, and 50 .mu.L of wash buffer containing
1:1000 dilution of streptavidin-alkaline phosphate conjugates
(Clontech, Palo Alto, Calif.) were added to each well. Each well
was incubated at room temperature for 30 min, and then washed three
times with wash buffer. Finally 100 .mu.L of AttoPhos (JBL
Scientific, San Luis Obispo, Calif.) was added to each well and
incubated at room temperature for 15 min. Fluorescence was
determined in a CytoFluor 2300 (Millipore, Bedford, Mass.) at 430
nm excitation and 560 nm emission. In order to quantitate the
amount of mRNA from fluorescence intensity, rabbit globin mRNA was
used as a control as previously described (Tominaga K, et al., Clin
Chem 1996:1750-1757, 1996). As shown in FIG. 9, captured mRNA from
10.sup.4 cells was approximately 5 ng from 5 different cell
lines.
[0105] In order to further analyze the potential degradation of
mRNA during hybridization, cytosolic fraction was collected after
hybridization, and treated with two rounds of
phenol/chloroform/isoamyl alcohol extraction followed by ethanol
precipitation. RNA was then analyzed by agarose gel
electrophoresis. As shown in FIG. 9 (right inset: Lane M, .lambda.
Hind III; lane 1, K562; lane 2, U937; lane 3, CaRI; lane 4, HepGII;
lane 5, KatolII; lane 6, CRL5800), 18s and 28s rRNA bands were
clearly present in all cells even after 1 hour incubation at room
temperature, suggesting that simple cytosolic fraction in the
presence of VRC was essentially free from RNase activity.
[0106] In conclusion, complete RT-PCR from starting cell suspension
can be conducted using just two plates; the glass fiber filter
plate and the oligo(dT)-immobilized polypropylene/polyolefine
plate. Furthermore, the 96-well format allows researchers to
conduct RT-PCR in high throughput fashion with potential full
automation. In this experiment, PCR products were analyzed by
agarose gel electrophoresis, however, PCR products may be
quantitated continuously by TaqMan system (Morris T, et al., J Clin
Microbiol 34:p2933-6, 1996). This experiment proved that this
system is a useful tool for high throughput RT-PCR.
[0107] It will be understood by those of skill in the art that
numerous and various modifications can be made without departing
from the spirit of the present invention. Therefore, it should be
clearly understood that the forms of the present invention are
illustrative only and are not intended to limit the scope of the
present invention.
Sequence CWU 1
1
6 1 21 DNA Artificial Sequence Primer cd4465 sense. 1 agtttcggag
cggatgaatg c 21 2 22 DNA Artificial Sequence Primer cd4465
antisense. 2 ggggcatcag aattttggtt ga 22 3 20 DNA Artificial
Sequence Primer for rabbit globin mRNA sense. 3 cgtggagagg
atgttcttgg 20 4 22 DNA Artificial Sequence Primer for rabbit globin
mRNA antisense. 4 aacgatattt ggaggtcagc ac 22 5 24 DNA Artificial
Sequence Primer for bcr-able sense. 5 gaccaactcg tgtgtgaaac tcca 24
6 23 DNA Artificial Sequence Primer for bcr-able antisense. 6
aaagtcagat gctactggcc gct 23
* * * * *