U.S. patent application number 11/389923 was filed with the patent office on 2006-07-27 for preservation of rna in a biological sample.
Invention is credited to Fredrik C. Kamme, Bernhard H. Meurers, Dmitri Talantov, Jingxue Yu.
Application Number | 20060166258 11/389923 |
Document ID | / |
Family ID | 33418220 |
Filed Date | 2006-07-27 |
United States Patent
Application |
20060166258 |
Kind Code |
A1 |
Kamme; Fredrik C. ; et
al. |
July 27, 2006 |
Preservation of RNA in a biological sample
Abstract
To preserve RNA in a biological sample for analysis, the sample
is incubated with an RNA preservative capable of precipitating RNA
in an aqueous solution, such as a triphenylmethane dye (e.g.,
methyl green, crystal violet, pararosaniline, or
tris-(4-aminophenyl)methane), cresyl violet, or cobalt ions. RNA
preservation may be used in an immunostaining assay and other
histochemical methods.
Inventors: |
Kamme; Fredrik C.; (San
Diego, CA) ; Meurers; Bernhard H.; (San Diego,
CA) ; Talantov; Dmitri; (San Diego, CA) ; Yu;
Jingxue; (San Diego, CA) |
Correspondence
Address: |
PHILIP S. JOHNSON;JOHNSON & JOHNSON
ONE JOHNSON & JOHNSON PLAZA
NEW BRUNSWICK
NJ
08933-7003
US
|
Family ID: |
33418220 |
Appl. No.: |
11/389923 |
Filed: |
March 27, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10826834 |
Apr 15, 2004 |
7056673 |
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11389923 |
Mar 27, 2006 |
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60465307 |
Apr 25, 2003 |
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Current U.S.
Class: |
435/6.12 ;
435/40.5; 435/6.14 |
Current CPC
Class: |
C12Q 1/6841 20130101;
C12N 15/1003 20130101; G01N 1/30 20130101 |
Class at
Publication: |
435/006 ;
435/040.5 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 1/30 20060101 G01N001/30; G01N 33/48 20060101
G01N033/48 |
Claims
1. (canceled)
2. (canceled)
3. (canceled)
4. (canceled)
5. A method of analyzing a biological sample comprising: (a)
contacting the biological sample with an RNA-preserving solution
comprising an aqueous solvent and an RNA preservative; (b)
incubating the biological sample with a buffer solution comprising
an aqueous buffered solvent and a binding agent capable of binding
to the biological sample; (c) detecting the binding agent bound to
the biological sample; and (d) identifying a target cell or tissue
within the biological sample based on the binding pattern of the
binding agent bound to the biological sample.
6. A method according to claim 5, wherein the biological sample
comprises a cell and the binding agent is a labeled molecule
selected from a group consisting of: an antibody capable of binding
to an antigen of the cell; a nucleic acid molecule capable of
hybridizing to a fragment of DNA of the cell under stringent
hybridization conditions; a nucleic acid molecule capable of
hybridizing to an mRNA of the cell under stringent hybridization
conditions; a lectin capable of binding to a carbohydrate-modified
substance of the cell; a substrate to an enzyme of the cell; and a
ligand capable of binding to a receptor of the cell.
7. A method according to claim 5, wherein the binding agent is a
compound labeled with a radio-isotope, a fluorescent molecule, or
biotin.
8. A method according to claim 5, wherein the RNA preservative is
selected from the group consisting of methyl green, crystal violet,
pararosaniline, tris-(4-aminophenyl)methane, cresyl violet, and
hexamine cobalt.
9. A method according to claim 5, further comprising: (e)
contacting the biological sample with a labeled nucleic acid
molecule capable of hybridizing to mRNA of the target cell or
tissue under stringent hybridization conditions; and (f) detecting
the labeled nucleic acid molecule bound to the target cell or
tissue.
10. A method according to claim 9, wherein the RNA preservative is
selected from the group consisting of triphenylmethane dyes, cresyl
violet, polyamines, and cobalt ions.
11. A method according to claim 5, further comprising: (e)
isolating the target cell or tissue from the biological sample; (f)
extracting mRNA from the isolated target cell or tissue; and (g)
analyzing the extracted mRNA by gene expression bioarray
analysis.
12. A method according to claim 11, wherein said isolating the
target cell or tissue from the biological sample comprises laser
capture microdissection.
13. A method according to claim 11, further comprising amplifying
the extracted mRNA from the isolated cell or tissue and labeling
the amplification product.
14. A method according to claim 11, further comprising contacting
the labeled amplification product with polynucleotide probes on a
microarray chip under hybridization conditions sufficient to
produce a hybridization pattern of complementary probe/target
complexes.
15. A method according to claim 11, further comprising
reverse-transcribing the extracted mRNA into cDNA.
16. A method according to claim 15, further comprising amplifying
the cDNA by a multiplex polynucleotide chain reaction and labeling
the amplification product.
17. A method according to claim 16, further comprising contacting
the labeled amplification product with polynucleotide probes on a
microarray chip under hybridization conditions sufficient to
produce a hybridization pattern of complementary probe/target
complexes.
18. A method according to claim 11, wherein the RNA preservative is
selected from the group consisting of triphenylmethane dyes, cresyl
violet, polyamines, and cobalt ions.
19. A method according to claim 11, wherein the RNA preservative is
a triphenylmethane dye selected from the group consisting of methyl
green, crystal violet, and pararosaniline.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods for preserving RNA
in a biological sample undergoing analysis. More particularly, the
present invention relates to methods of analyzing nucleic acid
expression patterns involving the preservation of RNA in biological
samples in histochemical assays.
BACKGROUND OF THE INVENTION
[0002] In the study of diseases, cell or tissue heterogeneity has
limited the information available from analysis of biological
samples. It has become increasingly important to be able to
investigate mRNA expression patterns within specific cell
populations at a specific physiological state.
[0003] Histochemical approaches have been applied to identify
specific cell populations within a biological sample. See, e.g.,
Okuducu et al. (2003), International Journal of Molecular Medicine
11:449-453. Such approaches include, e.g., techniques of
immunohistochemistry that detect proteins, in-situ hybridization
that measures messenger RNA, and fluorescence in-situ hybridization
(FISH) that detects changes in DNA. For example, histochemical
methods may be used to identify a cell type, e.g., microglia in the
brain as identified by the expression of complement receptor 3
(Graeber et al. (1988), J Neurosci Res 21:18-24), or to identify a
specific cell state, e.g., cellular activation stage as shown by
c-Fos expression (Sagar et al. (1988), Science 240:1328-1331).
[0004] Following the identification of specific cell populations at
a specific physiological state by histochemical analysis, mRNA
expression patterns of cells of interest can be analyzed by
traditional in-situ hybridization, which is often limited to
detecting the expression of only one or very few genes.
Alternatively, bioarray gene profiling can be performed. Cells of
interest can first be isolated via techniques such as laser capture
microdissection (LCM). mRNA can be extracted, amplified, and
reverse transcribed from the isolated cells. The resulting cDNAs
can be hybridized to a gene microarray chip. The resultant pattern
of hybridized nucleic acid provides information regarding the
genetic profile of the sample tested. This approach can be used to
examine the expression of multiple genes within individual cells or
tissues, and can be combined with other studies such as
electrophysiological, pharmacological and anatomical (retrograde
labeling) studies.
[0005] Analyses of gene expression patterns of an identified cell
or tissue type make it possible to directly correlate gene
expression with functional changes and lesion morphology at the
target cells or tissue. Results from such analyses can provide
important information on the effects of a drug within a biological
test system and help to elucidate mechanisms of drug-induced
toxicity and organ dysfunction, which are of great importance to
the field of drug discovery.
[0006] Unfortunately, RNA content has been shown to be severely
depleted during histochemical assays, for example, by
immunostaining of tissue sections (Fink et al. (2000), Lab Invest
80:327-333; Kohda et al. (2000), Kidney Int 57:321-331). This has
practically precluded mRNA expression analysis of immunostained
tissue, either by in situ hybridization or by microarray gene
profiling.
[0007] It was generally assumed that RNA in tissue sections was
degraded by endogenous RNases during the immunostaining protocol
(Murakami et al. (2000), Kidney Int 58:1346-1353). Therefore, to
preserve RNA in the tissue section during an immunostaining, large
amounts of RNase inhibitors (Murakami et al. (2000), supra) or
various tissue fixatives such as formalin (Fink et al. (2000),
supra) have been used in modified immunostaining protocols. See,
e.g., U.S. Patent Application Publication No. US 2002/0009768.
Although these protocols have had varying degrees of success, in
general they have to be extremely short in duration (Fend et al.
(1999), Am J Pathol 154:61-66). These modified immunostaining
protocols have limited usefulness because a longer incubation
period is required for the better sensitivity of immunostaining
detection.
[0008] Accordingly, a method to robustly preserve RNA in a
biological sample is needed to facilitate investigation of mRNA
expression patterns within a specific cell population or
tissue.
SUMMARY OF THE INVENTION
[0009] In one general aspect, the invention relates to a method of
analyzing a biological sample comprising: preserving RNA in the
biological sample by incubating the biological sample with an RNA
preservative in an aqueous solution so as to precipitate RNA;
histochemically staining the RNA-preserved biological sample;
histochemically analyzing the biological sample to identify
specific cell populations; and analyzing mRNA expression patterns
of the identified cells by a method comprising in-situ
hybridization, or isolating identified cells and subjecting the
isolated cells to bioarray gene profiling. In a preferred
embodiment, the histochemically analyzing comprises subjecting the
biological sample to a histochemical assay selected from: in situ
hybridization for detecting mRNA; fluorescence in-situ
hybridization for detecting DNA; immunocytochemistry assay for
detecting proteins; enzyme histochemistry assay for measuring
catalytic activities of enzymes; ligand-binding autoradiography for
studying receptor-ligand interactions; and glycohistochemistry
assay for detecting carbohydrate-modified substances.
[0010] In another general aspect, the invention pertains to a
method of analyzing a biological sample comprising: (a) contacting
the biological sample with an RNA-preserving solution comprising an
aqueous solvent and an RNA preservative; (b) incubating the
biological sample with a buffer solution comprising an aqueous
buffered solvent and a binding agent capable of binding to the
biological sample; (c) detecting the binding agent bound to the
biological sample; and (d) identifying a target cell or tissue
within the biological sample based on the binding pattern of the
binding agent bound to the biological sample. The method may
further comprise: (e) contacting the biological sample with a
labeled nucleic acid molecule capable of hybridizing to mRNA of the
target cell or tissue under stringent hybridization conditions; and
(f) detecting the labeled nucleic acid molecule bound to the target
cell or tissue. In an alternative preferred embodiment, the method
further comprises: (e) isolating the target cell or tissue from the
biological sample (e.g., using laser capture microdissection); (f)
extracting mRNA from the isolated target cell or tissue; and (g)
analyzing the extracted mRNA by gene expression bioarray
analysis.
[0011] In a preferred embodiments, the RNA preservative is selected
from triphenylmethane dyes (e.g., methyl green, crystal violet, and
pararosaniline), cresyl violet, polyamines, and cobalt ions.
[0012] Other aspects, features and advantages of the invention will
be apparent from the following disclosure, including the detailed
description of the invention and its preferred embodiments and the
appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1. This figure illustrates the accumulation of Neuron
Specific Enolase (NSE) mRNA in tissue incubation buffer during
immunostaining. The amount of NSE mRNA was measured as the amount
of cDNA via real time PCR, as shown on the Sybr green fluorescence
accumulation graphs. cDNA was synthesized from the tissue
incubation buffer PBS after tissue incubation (solid black line),
and before tissue incubation (dotted line). To control for
potential genomic DNA in the incubation buffer, PCR was also
performed without cDNA synthesis (interrupted line) from PBS after
tissue incubation. Two samples for each condition and two PCR
reactions for each sample were performed.
[0014] FIG. 2. This urea-agarose gel picture demonstrates the
precipitation of RNA from aqueous solution by
tris(4-aminophenyl)methane. RNA in each sample was analyzed by
urea-agarose gel analyses after dissolving the sample in urea gel
load buffer. The samples are, lane 1: RNA Size Standard; lane 2:
positive control, RNA from bacteriophage MS2; lane 3: RNA
precipitate which was formed when MS2 RNA was mixed with
tris(4-aminophenyl)methane, and was collected as the pellet after
centrifuging the mixture; lane 4: negative control 1, the presumed
"pellet" which was collected by centrifuging MS2 RNA only; lane 5:
negative control 2, the presumed "pellet" which was collected by
centrifuging tris(4-aminophenyl)methane only.
[0015] FIG. 3. This figure shows the preservation of RNA in tissue
sections by RNA preservatives during immunostaining. Rat brain
sections were immunostained, with--(RNA fix+immuno) or without
(immuno) the step of RNA preservation. At the end of
immunostaining, the tissue section was scraped off. RNA was
extracted from the scraped-off tissue sections, and was quantified
by RT-PCR for NSE mRNA. A) RNA preservation using methyl green. B)
RNA preservation using cresyl violet.
[0016] FIG. 4. This graph shows that RNA preservation decreased the
amount of RNA in the incubation buffer during immunostaining.
Tissue sections were treated with or without methyl green prior to
immunostaining. The amount of NSE mRNA in the tissue incubation
buffer after immunostaining was measured as the amount of cDNA by
RT-PCR. Units on Y-axis are arbitrary. Error bars indicate standard
deviation.
[0017] FIG. 5. This is a dendrogram of clustered microarray data.
The dendrogram indicates how closely related the conditions were
based on their gene expression profile. The most related conditions
were the two samples in the middle, one immunostained and one
non-stained, indicating that immunostaining with RNA fixation did
not alter the representation of mRNAs in the tissue sample.
DETAILED DESCRIPTION OF INVENTION AND ITS PREFERRED EMBODIMENTS
[0018] All publications cited below are hereby incorporated by
reference. Unless defined otherwise, all technical and scientific
terms used herein have the same meaning as commonly understood to
one of ordinary skill in the art to which this invention
pertains.
[0019] As used herein, the terms "comprising", "containing", and
"including" are used in their open, non-limiting sense.
[0020] A "biological sample" as used herein refers to a sample
containing or consisting of cell or tissue matter, such as cells or
biological fluids isolated from a subject. The subject can be a
eukaryotic organism, such as an animal, a plant, a worm, or a yeast
cell. Alternatively, the subject can be a prokaryotic organism,
such as a bacterial cell or an archaeal cell. Preferably, the
subject is a mammal, such as a rat, a mouse, a monkey, or a human,
who has been the object of treatment, observation or experiment.
Examples of biological samples include, for example, sputum, blood,
blood cells (e.g., white blood cells), amniotic fluid, plasma,
semen, bone marrow, tissue or fine-needle biopsy samples, urine,
peritoneal fluid, pleural fluid, and cell cultures. Biological
samples may also include sections of tissues such as frozen
sections taken for histological purposes.
[0021] In preferred embodiments, the biological sample is a
"clinical sample," which is a sample derived from a human patient.
A biological sample may also be referred to as a "patient sample."
A test biological sample is the biological sample that has been the
object of analysis, monitoring, or observation. A control
biological sample can be either a positive or a negative control
for the test biological sample. Often, the control biological
sample contains the same types of tissues, cells and biological
fluids as that of the test biological sample.
[0022] An "RNA preservative" as used herein refers to an agent that
is capable of precipitating RNA in an aqueous solution. Methods for
identifying RNA preservatives as well as examples of RNA
preservatives are described below.
[0023] A "histochemical assay" as used herein refers to a
biological assay useful for studying the biochemical composition of
tissues or cells by means of detecting a specific labeling that
correlates to a particular biochemical composition of the tissues
or cells. Such an assay is useful in identifying a particular cell
or tissue type based on studies of the biochemical composition of
tissues or cells. There are a variety of types-of histochemical
assays, including, for example, in situ hybridization for the
detection of mRNA, fluorescence in-situ hybridization (FISH) for
the detection of DNA, immunocytochemistry for the detection of
proteins, enzyme histochemistry (EH) for measuring the catalytic
activity of enzymes, ligand-binding autoradiography (LB) for the
study of receptor-ligand interactions, and glycohistochemistry for
the detection of carbohydrate-modified substances, e.g.,
glycoprotein.
[0024] An "in situ hybridization assay" is a biological assay that
histochemically detects a DNA or RNA sequence within cells or
tissues using labeled nucleic acid probes with base sequence
complementary to that of the target DNA or mRNA. Over the decades,
in situ hybridization has been used extensively to study the
distribution of mRNA species of particular genes within specific
compartments of a cell or tissue. Types of nucleic acid probes used
for in situ hybridization assay include single-stranded
oligonucleotides (usually 30-40 bases in length), either singly or
as cocktails, single-stranded RNA probes (riboprobes) about 300
bases long, or double-stranded cDNA sequences of various lengths.
Probes can be designed specifically against any known expressed
nucleic acid sequence. A number of different radioisotope and
non-isotopic labels are commercially available that may be used in
in-situ hybridization. For a review of known in-situ hybridization
methods, see McNicol et al. (1997), J. Pathol 182(3):250-61.
[0025] One exemplary in situ hybridization (ISH) assay involves:
fixing tissue with a formaldehyde solution, acetylating the tissue
with acetic anhydride in triethanolamine-HCl solution, dehydrating
the tissue with ethanol, and delipidating the tissue with
chloroform; incubating the tissue with radioactively or
fluorescently labeled nucleic acid probes in an aqueous buffer to
allow hybridization between the probes and their complementary mRNA
or DNA under stringent hybridization conditions; washing off the
unbound probes by an aqueous buffer; and detecting the probes that
bind to the tissue by autoradiography. "Stringent hybridization
conditions" has the meaning known in the art, as described in
Sambrook et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
(1989). Exemplary high stringency or stringent hybridization
conditions include, e.g.: 50% formamide, 5.times.SSC and 1% SDS
incubated at 42.degree. C., or 5.times.SSC and 1% SDS incubated at
65.degree. C., with a wash in 0.2.times.SSC and 0.1% SDS at
65.degree. C.
[0026] "Fluorescence in situ hybridization" or "FISH" as used
herein refers to a type of in situ hybridization which involves
hybridization of chromosome-specific, fluorescently labeled DNA
probes to target sequences, so that the target sequences are
stained or painted with fluorescent dyes and their chromosomal
locations and sizes can be determined using fluorescence
microscopy. The DNA probes can be for the whole chromosome or
centromere, or locus-specific. The use of variable FISH techniques
enhances the thorough interpretation of numerical and complex
chromosome aberrations, bridging the gap between conventional
chromosome banding analysis and molecular genetic DNA studies. This
staining is sufficiently distinct that the hybridization signal can
be seen both in metaphase spreads and in interphase nuclei (see,
e.g., review by Jiang et al.(2000), Diagn Mol Pathol
11(1):47-57).
[0027] An "immunohistochemistry assay" or "immunostaining assay" is
a biological assay that histochemically localizes immunoreactive
substances within cells or tissues using antibodies. The
immunoreactive substances can be any biological material that can
serve as an antigen and elicit an immune response. Exemplary
immunoreactive substances are proteins or small peptide haptens.
Primary antibodies may be monoclonal or polyclonal in origin.
Various primary antibodies are available commercially and through
specialist laboratories. Also, antibodies may be directed against
synthetic peptide sequences within a relatively short time scale,
enabling a greater degree of flexibility for studying new targets
of interest. A number of complete assay kits are also available in
which all reagents necessary for the immunohistochemical detection
of specific protein targets are included, usually with an optimized
protocol.
[0028] For a review of immunohistochemical methods, see Swanson
(1988), Am J Clin Pathol 90(3):333-9. An exemplary
immunohistochemistry assay involves: fixing a biological sample in
a fixative (e.g., acetone, alcohol, formalin, or paraformaldehyde);
incubating the sample with a primary antibody in an aqueous
solution to allow specific binding of the antibody to an antigen
within the sample; washing off the unbound antibody; contacting the
sample with a labeled secondary antibody or other agent (such as
bacterial protein A) to allow specific binding of the secondary
antibody or other agent to the primary antibody; washing off the
unbound secondary antibody or other agent; and detecting the amount
of labeled secondary antibody or other agent remaining with the
sample. The detection step may be done by chromogenic
detection--e.g. the secondary antibody is labelled with an enzyme
such as horseradish peroxidase or alkaline phosphatase, which is
detected using an enzyme substrate such as 3,3'-diaminobenzidineor
nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate
(toluidine salt), respectively. This gives a colored precipitate
that is visible under light microscopy.
[0029] An "enzyme histochemistry assay" is a biological assay that
histochemically measures the catalytic activity of enzymes within
cells or tissues using a detectable substrate for the enzyme. For a
review of enzyme histochemistry assays, see Boonacker et al.
(2001), J. Histochem Cytochem, 49(12):1473-86. Exemplary enzymes
are proteases. In some embodiments, the substrate is radioactively
labeled, chromogenic, or fluorogenic.
[0030] A "ligand-binding autoradiography" is a biological assay
that histochemically measures receptor-ligand interactions using a
labeled ligand. The interaction sites are localized through
detection of the labeled ligand. For a review, see Sovago (2001),
Brain Res Brain Res Rev 38(1-2):149-64.
[0031] A "glycohistochemistry assay" or "carbohydrate
histochemistry assay" is a biological assay that histochemically
localizes carbohydrate-modified substances, within cells or
tissues. For a review, see Danguy (1995), Eur J Histochem
39(1):5-14. An exemplary carbohydrate-modified substance is a
glycoprotein. The oligosaccharide modified glycoproteins have been
studied by classical histochemical techniques such as PAS, alcian
blue, and HID. More recently, lectins, a class of proteins that
have specific binding sites for specific mono- or
oligo-saccharides, have been applied in carbohydrate histochemistry
to study tissue or intracellular localizations of carbohydrate
residues. Lectins were originally obtained from the seeds of
leguminous plants, and were since found in many other plant and
animal sources. Different lectins that bind specifically to
different monosaccharides or glycans have been identified (fucose,
galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine,
N-acetylneuraminic acid and heparin). Lectins such as concanavalin
A and wheat germ agglutinin are used as analytical and preparative
agents in the study of glycoproteins.
[0032] An exemplary glycohistochemistry assay involves: fixing a
biological sample in a fixative (e.g., formaldehyde); incubating
the sample with a labeled lectin in an aqueous solution to allow
binding of the lectin to carbohydrate residues within the sample;
washing off the unbound lectin; and detecting the amount of labeled
lectin remaining with the sample, such by chromogenic detection
(see above).
[0033] The term "labeled", with regard to a labeled agent used in a
histochemical assay such as a nucleic acid probe, antibody, lectin,
or enzyme substrate, is intended to encompass direct labeling of
the agent by coupling (i.e., physically linking) a detectable
substance to the agent as well as indirect labeling of the agent
with another reagent that is directly labeled.
[0034] Labels that are directly detectable include fluorescent
labels and radioactive isotopes. Illustrative radioactive isotope
labels include, e.g., .sup.35S, .sup.32P, and .sup.3H. Preferred
fluorescers are those absorbing light in wavelengths of from about
300 to 900 nm, more preferably from about 400 to 800 nm, and where
the absorbance maximum is preferably at a wavelength ranging from
about 500 to 800 nm. Exemplary fluorescers that may be used in
singly labeled primers include fluorescein, rhodamine, BODIPY,
cyanine dyes and the like. Fluorescers are further described in
Smith et al., Nature (1986), 321: 647-679. Examples of indirect
labeling include detection of a primary antibody using a
fluorescently labeled secondary antibody, and end-labeling of a DNA
probe with biotin such that it can be detected with fluorescently
labeled streptavidin and the like.
[0035] "End-labeled" with regard to a labeled nucleic acid molecule
means that the label moiety is present at a region at least
proximal to the terminus. Preferred end labels have the moiety at
the 5' terminus of the nucleic acid molecule. The labeling can also
be at the 3' terminus, using for example the enzyme terminal
deoxynucleotidyl transferase.
[0036] "Laser capture microdissection (LCM)" refers to a technique
wherein a specimen is visualized under a microscope and then
overlaid with a layer of transfer material, such as a transparent
film, which when activated by a laser adheres to and extracts out
specific targeted elements within the specimen for further
processing. See, e.g., Emmert-Buck et al. (1996), Science,
274:998-1001. LCM as used herein also refers to a technique
wherein, under a microscope, regions of interest in a section are
outlined using a laser beam that cuts through the tissue. The
region of interest is then collected, either by laser pressure
catapulting (see, e.g., Schutze and Lahr (1998), Nat Biotechnol
16:737-742.), by the force of gravity, or by being attached to a
membrane that is separated from the section. Preferably, the
extracted specific target elements are individual cells or tissues
within complex tissues. The extracted cells or tissues may be
placed directly into DNA, RNA, or protein-extraction buffer for
processing.
[0037] LCM has been used to extract mRNA from frozen tissues after
a rapid immunostaining method of frozen sections (see Krizman et
al. (1996), Cancer Res, 56: 5380-5383). The method allows for an
ultra-specific LCM of frozen tissues. This technique may be useful
to analyze specific cell subtypes, such as basal cells in prostate
or various subcomponents of an inflammatory infiltrate (e.g., T- or
B-cells). This technique may also be useful in the identification
and isolation of cells from a similar population differing by their
metabolic state. For example, PCA cells that are proliferative may
be determined by Ki-67 (MIB-1) immunostaining. This technique may
also be useful in identifying cells that are morphologically
difficult to identify using standard LCM protocols. However, the
rapid immunostaining method may limit the sensitivity and
usefulness of this technique, as many immunostaining protocols
require longer incubation periods.
[0038] Methods on LCM analysis are known to those skilled in the
art. References on such methods can be found from literature (for
example, see Emmert-Buck et al. (1996), supra; Krizman et al.
(1996), supra); and U.S. Pat. No. 6,420,132, which discloses a
method and apparatus for microdissection of targets within tissue
or other specimen samples smaller than approximately 10 microns in
diameter).
[0039] "Bioarrays" as used herein refers to a substrate, e.g., a
substantially planar substrate such as a biochip or gene chip,
having a plurality of polymeric molecules spatially distributed
over, and stably associated with or immobilized on, the surface of
the substrate. Bioarrays of both polypeptides and polynucleotides
have been developed and found use in a variety of bioarray
applications, such as screening and DNA sequencing and gene
expression analysis.
[0040] "Gene expression bioarray analysis" refers to an assay
wherein a bioarray of "probe" oligonucleotides is contacted with a
nucleic acid sample of interest, e.g., a target sample, such as
poly A mRNA from a particular tissue type, or a reverse transcript
thereof. See, e.g., Nees et al. (2001), Curr Cancer Drug Targets,
1(2):155-75. Contact is carried out under hybridization conditions
and unbound nucleic acid is removed. The resultant pattern of
hybridized nucleic acid provides information regarding the genetic
profile of the sample tested. Gene expression analysis can measure
expression of thousands of genes simultaneously, providing
extensive information on gene interaction and function. Gene
expression analysis may find use in various applications, e.g.,
identifying expression of genes, correlating gene expression to a
particular phenotype, screening for disease predisposition, and
identifying the effect of a particular agent on cellular gene
expression, such as in toxicity testing.
[0041] Exemplary bioarray formats include oligonucleotide arrays,
spotted arrays, microarrays (an array that is miniaturized so as to
require microscopic examination for visual evaluation), and
macroarrays (an array that is large enough to permit visual
evaluation without the aid of a microscope). Methods on gene
expression bioarray analysis are known to those skilled in the art.
See, e.g., review by Yang et al. (2002), Nat Rev Genet 3(8):
579-88), and U.S. Pat. No. 6,004,755, which discloses methods on
quantitative gene expression analysis using a DNA microarray.
[0042] In performing histochemical assays a conventional fixative
may function by chemically introducing cross-links between
molecules in the sample, for example by using glutaraldehyde. The
fixative may also function by precipitating molecules in the
sample, for example, by using ethanol or acetone. For a discussion
of fixatives, see U.S. Patent Application Publication No. US
2002/0009768 Al. However, chemical cross-linking is undesirable for
preserving RNA in the tissue, as it may impede the final extraction
of RNA or even break the RNA strand (Goldsworthy et al. (1999), Mol
Carcinog 25:86-91; Kohda et al. (2000), Kidney Int 57:321-331).
Alcohol precipitation does not prevent the RNA precipitates from
dissolving in subsequent steps involving aqueous buffers.
[0043] The present invention provides a method for preserving RNA
in a biological sample during a histochemical assay wherein the
biological sample is preserved with an RNA preservative that
precipitates RNA in an aqueous solution. The RNA preservatives that
can be used in the method of the invention precipitate RNA in
aqueous buffer solutions.
[0044] The selection of a suitable RNA preservative is within the
purview of those of ordinary skill in the art. For example, an RNA
preservative can be identified using a method comprising: 1)
contacting RNA molecules in an aqueous solution, such as water,
containing buffer in which the testing compound or control is
dissolved; and 2) comparing the amount of RNA precipitates in the
solution containing the testing compound with that of the control.
The testing compound that is capable of precipitating RNA in an
aqueous solution would result in significantly more RNA
precipitation than that of the control. The amount of RNA
precipitation in the identification assay can be measured by
methods known to those skilled in the art. An example of using such
method is illustrated in Example 2 below, wherein RNA molecules in
water were first contacted with a testing compound, RNA
precipitates were then isolated by centrifugation, and the amount
of isolated RNA precipitates was quantified by gel electrophoresis.
Alternatively, the amount of RNA precipitates can be measured by
light scattering in the aqueous solution following a procedure
similar to that described previously for studying DNA condensation
by polyamines (Vijayanathan et al. (2001), Biochemistry
40:13644-13651).
[0045] It has been described that triphenylmethane compounds, such
as methyl green, bind double-stranded nucleic acids (Adams (1968),
J Pharm Pharmacol 20:Suppl:18S+; Armstrong and Panzer (1972), J Am
Chem Soc 94:7650-7653; Muller and Gautier (1975), Eur J Biochem
54:385-394; Melnick and Pickering (1988), Biochem Int 16:69-75; Fox
et al. (1992), Eur J Histochem 36:263-270; and Kim and Norden
(1993), FEBS Lett 315:61-64). Using assays described supra, it was
found that triphenylmethane dyes such as methyl green, crystal
violet and pararosaniline precipitate RNA. It was also found that
cresyl violet, which belongs to a different chemical class,
precipitates RNA as well. In addition, cobalt ions were also found
capable of precipitating RNA. Thus, compounds from different
chemical classes and even ions are capable of precipitating RNA
from aqueous solutions.
[0046] Preferably, the RNA preservatives used in the method of the
invention precipitate RNA in aqueous buffer solutions, but do not
interfere with the subsequent RNA extraction or RNA analysis. More
preferably, the RNA preservatives precipitate RNA in aqueous buffer
solutions, but do not interfere with the subsequent RNA extraction
or RNA analysis, and are compatible with immunohistochemistry or
other types of histochemical assays. For example, preferred RNA
preservatives do not interfere with antigen-antibody interaction,
and do not impart a color to the biological sample that would mask
the chromogen used for immunostaining. Examples of RNA
preservatives that can be used in the method of the invention
include triphenylmethane dyes (such as methyl green, crystal
violet, pararosaniline, or tris-(4-aminophenyl)methane), cresyl
violet, polyamines, and cobalt ions.
[0047] Exemplary polyamine RNA preservatives include spermine,
spermidine, 1,10-diamino-4,7-diazadecane,
1,11-diamino-4,8-diazaundecane, 1,13-diamino-4,10-diazatridecane,
1,14-diamino-4,11-diazatetradecane,
1,15-diamino-4,12-diazapentadecane,
1,16-diamino-4,13-diazahexadecane,
1,17-diamino-4,14-diazaheptadecane,
1,18-diamino-4,15-diazanonadecane, 1,19-diamino-4,16-diazaeicosane,
and 1,20-diamino-4,17-diazaheneicosane.
[0048] Because RNA is preserved in the biological sample during the
assay, mRNA expression patterns of a particular cell or tissue may
now be analyzed after their identification by the histochemical
assay. Thus, in one aspect, the present invention relates to a
method of analyzing expression of an mRNA molecule at a target cell
or tissue, comprising: contacting a biological sample with an
RNA-preserving solution comprising an aqueous solvent and an RNA
preservative; incubating the biological sample with an incubation
buffer comprising a buffered aqueous solution and an agent capable
of binding to the biological sample; detecting the binding agent
bound to the biological sample; identifying target cell or tissue
within the biological sample based on the binding pattern of the
binding agent bound to the biological sample; contacting the
biological sample with a labeled nucleic acid molecule capable of
hybridizing to the mRNA of the target cell or tissue under
stringent hybridization conditions; and detecting the labeled
nucleic acid molecule bound to the target cell or tissue.
[0049] Preferably, the contacting of the biological sample with an
RNA-preserving solution is performed before exposing the biological
sample to the aqueous incubation buffer. The method may further
comprise fixing the biological sample in a fixative, such as
acetone, alcohol, formalin, or glutaraldehyde, with the RNA
preservation being performed after the fixing.
[0050] The type of histochemical assay can involve, for example, in
situ hybridization, FISH, immunohistochemistry, enzyme
histochemistry, ligand-binding autoradiography, or
glycohistochemistry. Accordingly, binding agents that may be used
include, for example, a labeled complementary nucleic acid probe,
an antibody, a labeled enzyme substrate, a labeled ligand, or a
labeled lectin. The contacting of the biological sample with an
agent and the detecting of the agent that binds to the biological
sample may be conducted using techniques known in-the art. An
exemplary immunohistochemistry assay is described in Example 3
below.
[0051] The invention also provides a method of analyzing expression
of multiple mRNA molecules at a target cell or tissue within a
biological sample, comprising: contacting a biological sample with
an RNA-preserving solution comprising an aqueous solvent and an RNA
preservative; incubating the biological sample with an incubation
buffer comprising a buffered aqueous solution and an agent capable
of binding to the tissue or cell matter in the biological sample;
detecting the binding agent bound to the biological sample;
identifying the target cell or tissue within the biological sample
based on the binding pattern of the agent bound to the biological
sample; isolating the target cell or tissue from the biological
sample; extracting RNA from the isolated target cell or tissue; and
analyzing the extracted mRNA by gene expression bioarray
analysis.
[0052] Any one or a combination of the histochemical analyses
techniques described herein can be used to identify the target cell
or tissue. For example, identification of the target cell or tissue
can be based on a specific gene expression pattern revealed by an
in situ hybridization assay, a specific protein expression pattern
revealed by an immunohistochemistry assay, and/or a specific
glycoprotein expression pattern revealed by a glycohistochemistry
assay.
[0053] In preferred embodiments, two types of assays are performed
to analyze mRNA expression patterns at the identified target cell
or tissue after histochemical analysis. In one embodiment, in situ
hybridization quantifies the mRNA expression level of a particular
gene within the target cell or tissue. The measured mRNA expression
level is compared with that of the protein expression level from
the previous immunochemistry assay, the mRNA expression level of a
different gene from the previous in situ hybridization assay, or
the enzymatic activity from the previous enzyme histochemistry
assay.
[0054] Previously, histochemical analysis combining immunostaining
with in situ hybridization had been carried out to study the
correlation of protein expression and mRNA expression (see, for
example, Quan et al. (1997), Proc. Natl. Acad. Sci. USA, 94:
10985-10990). However, because these previous assays adopted the
rapid immunostaining protocol in avoiding loss of RNA during the
incubation, they had limited detection sensitivity and usefulness.
In the method of the present invention, pre-treatment of the
biological sample with an RNA preservative precipitates RNA within
the sample. RNA molecules are not lost during the incubation and
washing steps of immunostaining. Thus, higher protein detection
sensitivity may now be obtained by incubating the biological sample
in aqueous solution as long as needed during immunostaining while
still maintaining high sensitivity for RNA detection after the
immunostaining.
[0055] In another embodiment, mRNA expression patterns of multiple
genes within the identified target cell or tissue are analyzed by
first isolating the identified cell or tissue from the biological
sample and then analyzing the extracted RNA by gene expression
bioarray analysis. Such a method allows gene profiling on a
particular cell, cell type, and tissue.
[0056] Target cell or tissue matter can be isolated from the
biological sample by several approaches. In the past, this has been
done through microdissection with 30-gauge needles (Vocke, et al.
(1996), Cancer Res 56: 2411-2416). Recent advances in
microdissection techniques include manual techniques, laser
microdissection, laser capture microdissection (LCM), and laser
catapulting (Eltoum et al. (2002), Adv Anat Pathol,
9(5):316-22).
[0057] LCM is effective in the molecular analysis of complex
tissues because it combines the topographic precision of microscopy
with the power of molecular genetics, genomics, and proteomics. As
described above, LCM is a technique that is capable of isolating
individual cells or tissues from complex tissues using laser
capture microdissection. Laser capture microdissection occurs where
the transfer polymer film is placed on a substrate overlying
visualized and selected cellular material from a sample for
extraction. The transfer polymer film is focally activated (melted)
with a pulse brief enough to allow the melted volume to be confined
to that polymer directly irradiated. Methods of LCM, such as
contact LCM or non-contact LCM, using either condenser-side (or
beam passage through polymer before tissue) or epi-irradiation (or
laser passage through tissue before polymer), and other LCM
techniques can be used in the present invention. Example 4
illustrates the use of an exemplary LCM technique.
[0058] In obtaining the sample of RNA to be analyzed from the
isolated cell or tissue, the isolated cell or tissue may be
subjected to a number of different processing steps. For example,
such processing steps might include tissue homogenization, cell
isolation and cytoplasmic extraction, nucleic acid extraction and
the like, or other suitable processing steps known in the art.
Techniques for isolating RNA from cells, tissues, organs or whole
organisms are known in the art; see, e.g., Maniatis et al.,
Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press),
1989. Kits that are commercially available can also be used to
extract RNA from a biological sample following the instructions
provided by the manufacturer of the kit.
[0059] Standard hybridization and detection protocols are
insufficient for micro- to milli-gram quantities of tissue or
cells, such as those obtained from LCM. Amplification systems
utilizing T7 RNA polymerase can provide multiple CRNA copies from
mRNA transcripts, permitting microarray studies with reduced sample
inputs. For example, an optimized T7-based amplification system for
microarray analysis that yields between 200- and 700-fold
amplification has been described by Pabon et al. ((2001)
Biotechniques 31(4):874-9).
[0060] The amplified cRNA or aRNA samples can then be reverse
transcribed into target nucleic acid (cDNA), for example by priming
with random hexamers under conditions sufficient for enzymatic
extension of the hybridized primer.
[0061] The reverse transcribed cDNA can be labeled by using labeled
primers. The label may be attached to one or more of the
nucleotides in the primer, either directly or through a linking
group, using methodology known in the art. In a preferred
embodiment in which the label is biotin, the number of biotinylated
dNTPs in the primer will be at least 1 and may be as high as 12,
but will preferably be about 7. Preferably, the cDNA can be labeled
by direct incorporation of fluorescently labeled DNTP, for example
Cy3-dCTP, during reverse transcription of the amplified aRNA.
[0062] In preparing the end-labeled target nucleic acid (cDNA), the
primer is contacted with the mRNA or cRNA with a reverse
transcriptase and other reagents necessary for primer extension
under conditions sufficient for first-strand cDNA synthesis.
Exemplary primer-extension reagents include: dNTPs; buffering
agents, e.g. Tris.Cl; cationic sources, both monovalent and
divalent, e.g. KCl and MgCl.sub.2; RNAase inhibitor and sulfhydril
reagents, e.g. dithiothreitol; and the like. A variety of enzymes,
such as DNA polymerases, possessing reverse transcriptase activity
can be used for the first-strand cDNA synthesis. Examples of
suitable DNA polymerases include the DNA polymerases derived from
organisms selected from thermophilic bacteria and archaebacteria,
retroviruses, yeasts, Neurosporas, Drosophilas, primates and
rodents. Preferably, the DNA polymerase is selected from Moloney
murine leukemia virus (M-MLV) (described in U.S. Pat. No.
4,943,531) and M-MLV reverse transcriptase lacking RNaseH activity
(described in U.S. Pat. No. 5,405,776), human T-cell leukemia virus
type I (HTLV-I), bovine leukemia virus (BLV), Rous sarcoma virus
(RSV), human immunodeficiency virus (HIV) and Thermus aquaticus
(Taq) or Thermus thermophilus (Tth) (described in U.S. Pat. No.
5,322,770), avian myeloblastosis virus reverse transcriptase, and
the like. Suitable DNA polymerases possessing reverse transcriptase
activity may be isolated from an organism, obtained commercially or
obtained from cells which express high levels of cloned genes
encoding the polymerases by methods known to those of skill in the
art. The particular manner of obtaining the polymerase preferably
is chosen based on factors such as convenience, cost, availability
and the like. Of particular interest because of their commercial
availability and well characterized properties are avian
myeloblastosis virus reverse transcriptase and M-MLV.
[0063] The order in which the reagents are combined in performing
reverse transcription may be modified as desired. One protocol that
may be used involves the combination of all reagents, except for
the reverse transcriptase on ice, followed by addition of the
reverse transcriptase and mixing at around 4.degree. C. After
mixing, the temperature of the reaction mixture is raised to
37.degree. C. followed by incubation for a period of time
sufficient for first-strand cDNA primer extension product to form,
e.g., about 1 hour.
[0064] The reverse transcripts (cDNA) from the reverse
transcription can be further amplified by polymerase chain reaction
following standard protocols known to those skilled in the art.
[0065] The extracted mRNA can be reverse transcribed and amplified
by multiplex quantitative reverse transcription-polymerase chain
reaction (RT-PCR), a form of RT-PCR that involves the simultaneous
amplification of more than one reverse transcript of a target mRNA
per reaction by mixing multiple primer pairs with different
specificities. The labeled cDNA that has been reverse transcribed
from target mRNA nucleic acids is contacted with an array of
polynucleotide probes stably associated with the surface of a
substantially planar solid support (chip) under hybridization
conditions sufficient to produce a hybridization pattern of
complementary probe/target complexes. A variety of different arrays
known in the art may be used in the present invention.
[0066] The polymeric or probe molecules of the arrays which are
capable of sequence specific hybridization with target nucleic acid
may be polynucleotides or hybridizing analogues or mimetics
thereof, for example: nucleic acids in which the phosphodiester
linkage has been replaced with a substitute linkage, such as
phosphorothioate, methylimino, methylphosphonate, phosphoramidate,
guanidine and the like; nucleic acids in which the ribose subunit
has been substituted, e.g., hexose phosphodiester; peptide nucleic
acids; and the like. The length of the probes stably associated
with the chip will generally range from 10 to 1000 nts. In some
embodiments, the probes are oligonucleotides ranging from 15 to 150
nts, preferably from 15 to 100 nts in length. In other embodiments
the probes are longer, ranging in length from 150 to 1000 nts,
where the polynucleotide probes may be single- or double- stranded,
preferably double-stranded, and may be PCR fragments amplified from
cDNA.
[0067] The probe molecules on the surface of the substrates
preferably correspond to known genes of the physiological source
being analyzed and are positioned on the array at a known location
so that positive hybridization events may be correlated to
expression of a particular gene in the physiological source from
which the target nucleic acid sample is derived. Because of the
manner in which the target nucleic acid sample is generated, as
described below, the arrays of probes will preferably have
sequences that are complementary to the non-template strands of the
gene to which they correspond.
[0068] The substrates with which the probe molecules are stably
associated may be fabricated from a variety of materials, such as
plastics, ceramics, metals, gels, membranes, glasses, and the like.
The arrays may be produced according to any convenient methodology,
such as preforming the probes and then stably associating them with
the surface of the support or growing the probes directly on the
support. A number of different array configurations and methods for
their production and usage are known in the art. See, e.g., U.S.
Pat. Nos. 5,445,934; 5,532,128; 5,556,752; 5,242,974; 5,384,261;
5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,472,672;
5,527,681; 5,529,756; 5,545, 531; 5,554,501; 5,561,071; 5,571,639;
5,593,839; 5,599,695; 5,624,711; 5,658,734; and 5,700,637.
[0069] In embodiments of assays of the subject invention, the
labeled target nucleic acid is contacted with the array under
conditions sufficient for hybridization of target nucleic acid to
probe-to occur. Suitable hybridization conditions are well known to
those of skill in the art and reviewed in Maniatis et al, supra,
and WO 9521944. The conditions can be modified to achieve a desired
specificity in hybridization, e.g. highly stringent or moderately
stringent conditions. For example, low stringency hybridization
conditions may be at 50.degree. C. and 6.times.SSC (0.9 M sodium
chloride/0.09 M sodium citrate), while hybridization under
stringent conditions may be at 50.degree. C. or higher and
0.1.times.SSC (15 mM sodium chloride/0.15 mM sodium citrate).
[0070] Following hybridization, where non-hybridized labeled
nucleic acid is capable of emitting a signal during the detection
step, a washing step is employed where unhybridized labeled nucleic
acid is removed from the support surface, generating a pattern of
hybridized nucleic acid on the substrate surface. A variety of wash
solutions and protocols for their use are known to those of skill
in the art and may be used. Example 5 illustrates a procedure for
performing gene profiling using microarray analysis.
[0071] The resultant hybridization pattern(s) of labeled nucleic
acids may be visualized or detected in a variety of ways. The
particular manner of detection is preferably chosen based on the
particular label of the nucleic acid, and exemplary detection
techniques include scintillation counting, autoradiography,
fluorescence measurement, calorimetric measurement, light emission
measurement and the like.
[0072] To further illustrate the invention, the following examples
are provided.
EXAMPLE 1
RNA Diffusion Into the Tissue Incubation Buffer During
Immunostaining
[0073] To characterize the mechanism by which RNA is lost from the
tissue sections during immunostaining, RNA from immunostained
tissue sections was extracted and compared to that from tissue
sections that had not undergone immunostaining. Frozen sections
were air-dried and fixed in cold acetone for 2 min. After a quick
rinse in phosphate buffered saline (0.137 M NaCl, 0.0027 M KCl,
0.01 M phosphate buffer pH 7.4, PBS), sections were incubated in
methyl green solution (Vector, Burlingame, Calif.) for 2 min. They
were then rinsed in PBS and incubated in OX-42 antibody (Serotec,
Raleigh, N.C.) diluted 1:100 in PBS with 0.5% acetylated BSA
(Sigma) for 5 min. Sections were rinsed in PBS and then incubated
in biotinylated goat anti-mouse antibody (Chemicon, Temecula,
Calif.) diluted 1:100 in PBS with 0.5% acetylated BSA for 5 min.
After rinsing in PBS, sections were incubated in 0.1 M Tris-HCl, pH
8.0 for 1 min and then transferred to PBS with alkaline
phosphatase-streptavidin (Vector), diluted 1:100, and incubated for
5 min. Sections were then rinsed in PBS and colour was developed
for 10 min using fuchsin red (Dako, Carpinteria, Calif.) as the
substrate. Sections were rinsed in RNase-free water and quickly
counterstained with Mayer's Hematoxylin. After subsequent washes in
PBS and water, the sections were dehydrated through increasing
concentrations of ethanol and finally xylene. Sections were left to
air dry. Sections were scraped off from the slide into a 1.5-ml
Eppendorf tube and RNA was extracted using the Rneasy kit (Qiagen,
Alameda, Calif.) according to the manufacturer's protocol. For
RT-PCR, 10 .mu.l of the extracted RNA (from a total volume of 30
.mu.l) was mixed with 1 .mu.g oligo-dT primer (Operon, Alameda,
Calif.) and heated to 70.degree. C. for 10 minutes (min), and then
cooled on ice. cDNA was synthesized using Superscript II
(200u/reaction) (Invitrogen, Carlsbad, Calif.) in 50 mM Tris-HCl,
75 mM KCl, 3 mM MgCl.sub.2, 20 mM DTT, 500 .mu.M dNTPs and 40 units
of RNase Block (Stratagene, La Jolla, Calif.) in a 20-.mu.l
reaction for 2 hours at 42.degree. C. The reaction was terminated
by incubating at 70.degree. C. for 10 min. Real time quantitative
PCR was performed using a Smartcycler (Cepheid, Sunnyvale, Calif.).
An aliquot of the cDNA was removed and diluted 4-fold. Two gl of
the dilution was used for real time PCR analysis. The mix contained
2 U ExTaq (Panvera, Madison, Wis.), 0.2.times. SybrGreen (Molecular
Probes, Eugene, Oreg.), 0.4 .mu.M of each primer, 200 mM dNTPs
(Amersham, Piscataway, N.J.), 2-4 mM MgCl.sub.2 (depending on
primers), 0.12 mg/ml BSA (Sigma), 90 mM trehalose (Sigma) and 0.12%
Tween 20 (Sigma) in 10 mM Tris-HCl pH 8. The PCR parameters were
95.degree. C./90 sec, 40 cycles of 95.degree. C./5 sec,
54-70.degree. C. (depending on primers)/7 sec and 72.degree. C./15
sec. At the end of each program a melt curve analysis was done.
[0074] Fragmentation of RNA, whether by RNase activity, mechanical
shearing or chemical hydrolysis, cleaves the polynucleotide
internally at random locations (though different RNases will
preferentially cleave adjacent to certain bases). Increasing
fragmentation of RNA will result in progressively shorter strands
of cDNA produced by priming from the 3'-end of the RNA using an
oligo-dT primer. The length of cDNA product can be detected by real
time quantitative PCR directed towards different amplicons from the
5'-end and the 3'-end portions of the mRNA. The shorter the average
length of the population of individual cDNA molecules for a given
mRNA, the more biased the cDNA will be towards the 3'-end and the
lower the 5'-end/3'-end ratio will by as measured by PCR.
[0075] To detect fragmentation of RNA in the tissue section during
immunohistochemistry, two pairs of primers were designed for rat
neuron specific enolase (NSE, GenBank DNA Accession No: AF019973,
2222 bases in total). One primer pair directed to the 5'-end
portion of the mRNA (bases 184-474 of the NSE mRNA), SEQ ID NO:
1,5'-CCTCCACTGGCATCTATGAG-3' and SEQ ID NO:
2,5'-CCTCTATCGCCACATTGCTC-3'; and the other directed to the 3'-end
portion of the mRNA (bases 1936-2221 of the NSE mRNA), SEQ ID NO:
3,5'-AGATGACCTAGGATGGGAGG-3' and SEQ ID NO:
4,5'-GTGTGCACTGTGATTCAGAC-3'.
[0076] The amount of RNA in immunostained tissue, judged by PCR
against the 3'-end portion of the NSE mRNA, was merely 6% of the
non-immunostained tissue. Thus 94% of NSE mRNA, and likely of total
mRNA as well, was lost during the immunostaining procedure.
However, the average ratios of 5'-end/3'-end PCR were nearly
identical for immunostained versus non-immunostained tissue: 8.74%
and 8.75% respectively. Thus, even though the majority of the RNA
content was gone from the tissue section, there were no signs of
RNA degradation. The conclusion was that RNA was lost during
immunohistochemistry not by degradation, but by some other
mechanism, likely diffusion into the solution.
[0077] To test the hypothesis that RNA was lost by diffusion into
the aqueous solution during immunostaining, the aqueous solution
was collected after the immunostaining procedure. cDNA was
synthesized from the phosphate buffer saline (PBS) solution that
was used in the immunostaining for tissue incubation, and PCR for
NSE cDNA corresponding to the 3' portion of the NSE mRNA (bases
1936-2221 of the NSE mRNA) was performed using real-time
quantitative PCR. As shown in FIG. 1, NSE mRNA is clearly present
in the solution after immunostaining, thereby confirming the
hypothesis that RNA dissolves in aqueous buffers during incubation
of the tissue section.
[0078] Thus, the primary cause for the loss of RNA during
immunohistochemistry is not RNA degradation by RNases, but
diffusion of the RNA into the aqueous assay buffer during
incubation. The main challenge in preserving RNA during
immunohistochemistry therefore is not to prevent RNase activity,
but to prevent the dissolution of RNA into the aqueous buffers
used.
EXAMPLE 2
Identification of RNA Preservatives
[0079] A testing compound, tris(4-aminophenyl)methane, was first
incubated with RNA molecules in an aqueous solution, such as water.
The mixture of the compound and RNA was then centrifuged at 10,000
g for 20 min to collect any precipitate. The collected precipitate
was then analyzed by denaturing agarose gel electrophoresis. A band
of the proper molecular weight (depending on the type of RNA used)
indicated that the compound precipitated RNA.
[0080] Other compounds that precipitated RNA from an aqueous buffer
included methyl green, cresyl violet, tris(4-aminophenyl)methane
and hexamine cobalt. Compounds that were found positive by this
screening assay were then tested for efficacy in preventing RNA
loss during immunohistochemistry on brain tissue sections.
EXAMPLE 3
Immunohistochemical Assay
(A) Comparison Example--OX42 Immunostaining Without RNA
Preservation
[0081] Frozen sections of tissues were air dried and fixed in cold
acetone for 2 min. Sections were rinsed in PBS and incubated with
OX42 antibody (Serotec, Raleigh, NC) diluted 1:100 in PBS with 0.5%
acetylated BSA (Sigma) for 5 min. Sections were then rinsed in PBS
and incubated with biotinylated goat-anti-mouse antibody (Chemicon,
Temecula, Calif.), diluted 1:100 in PBS with 0.5% acetylated BSA
for 5 min. Slides were again rinsed in PBS and then incubated 1 min
in 100 mM Tris-HCl, pH 8. Then, slides were incubated with
peroxidase-conjugated streptavidin (Jackson ImmunoResearch
Laboratories, West Grove, Pa.), which was diluted 1:100 in PBS, for
5 min. Sections were rinsed in PBS, and detection was performed
using AEC (DAKO, Carpinteria, Calif.) for 5 min. Sections were
rinsed in water and counterstained with Mayer's Hematoxylin
(BioGenex, San Ramon, Calif.) for 5 sec. Sections were rinsed in
PBS, then water and finally left to air dry.
(B) Inventive Example--OX42 Immunostaining with RNA
Preservation
[0082] The RNA-preserving step was inserted right after the acetone
fixation in the beginning of the staining protocol of Example 3(A)
above.
[0083] (i) Using methyl green as the RNA preservative, sections
were at this stage rinsed in PBS (Invitrogen, Carlsbad, Calif.) and
incubated in methyl green (Vector, Burlingame, Calif.) for 2 min.
The staining protocol described above was then followed.
[0084] (ii) Using cresyl violet as the RNA preservative, sections
were first fixed in 100% ethanol for 1 min (instead of acetone) and
incubated in 95% ethanol/10 sec, 70% ethanol/10 sec, 50% ethanol/10
sec, PBS/10 sec and 0.83% cresyl violet in H.sub.2O/40 sec.
Sections were then immunostained as described above, starting with
a PBS rinse and incubation in the primary antibody. After the final
PBS rinse, the Nissl stain was destained by successive incubations
in 70% ethanol/10 sec, 95% ethanol/10 sec, 95% ethanol+1.6% acetic
acid/5-10 sec, 95% ethanol/10 sec and finally 100% ethanol/10 sec.
If sections were used for laser capture, they were cleared by a 1
min incubation in xylene and then left to air dry.
(C) Inventive Example--IB4 Immunostaining with RNA Preservation
[0085] Frozen tissue sections were fixed in 100% ethanol for 1 min,
and then rinsed in 95% ethanol, 70% ethanol and PBS. The sections
were then stained in 0.83% cresyl violet in H.sub.2O for 45 sec and
rinsed in PBS. Sections were then incubated with 20 .mu.g/ml
peroxidase-conjugated IB4 (Sigma) in PBS with 1% acetylated BSA for
15 min. After a PBS rinse, sections were developed with DAB
(Vector) for 5 min. The reaction was stopped with a PBS rinse. The
Nissl stain was destained by successive rinses in 70% ethanol, 95%
ethanol, 95% ethanol+1.6% acetic acid, 100% ethanol and finally
cleared in xylene for 1 min and left to air dry.
(D) Quantification of RNA after Immunohistochemistry
[0086] To measure the RNA content in tissue sections after
immunohistochemistry, whole sections were scraped off into a 1.5 ml
centrifuge tube. RNA was quantified by RT-PCR for the 3' portion of
NSE following the procedures described in Example 1.
[0087] FIG. 3A shows the RNA preservation effect of methyl green on
NSE content in the tissue. After immunohistochemistry with and
without methyl green pretreatment, approximately 70% of the RNA, as
indicated by NSE, was lost after immunohistochemistry in the
absence of methyl green, whereas the inclusion of methyl green into
the assay protocol resulted in a high recovery of RNA. Thus, methyl
green prevented the loss of RNA during immunohistochemistry. FIG.
3B shows a similar experiment using cresyl violet, with a similar
result.
[0088] If these preservative compounds effectively prevent the
diffusion of RNA into the staining solution, then the amount of RNA
in the solution after the immunohistochemical protocol should be
reduced when these compounds are used. FIG. 4 shows that this is
indeed the case. Inclusion of methyl green significantly reduced
the amount of NSE in the tissue incubation solution.
[0089] To be desirable RNA preservatives, the compounds used should
be compatible with the particular immunohistochemistry assay being
performed. That entails, e.g., neither interfering with the
antigen-antibody interaction, nor imparting a color to the section
that would mask the chromogen used for immunostaining. After
immunostaining, sections were counterstained to visualize cell
nuclei. The observations reflected that pre-treating the tissue
section with methyl green did not affect the observed
immunostaining results.
EXAMPLE 4
Laser Capture Microdissection (LCM)
[0090] Cells were captured using the PixCell II laser capture
microdissection instrument (Arcturus, Mountain View, Calif.) onto
standard caps, TF100, following manufacture's instructions. Caps
were then put in 500-.mu.l tubes and frozen on dry ice. Cells were
picked from dorsal hippocampus Cal in coronal sections from adult
female Sprague-Dawley rats. One hundred cells were picked per
sample.
EXAMPLE 5
Gene Expression Profiling After Immunostaining
[0091] To test the efficacy of RNA preservation with gene
expression profiling, individual cells were isolated by laser
capture microdissection and were analyzed for gene expression by
microarray analysis. Brain sections were pretreated with an RNA
preservative, cresyl violet, immunostained with IB4, which stains
microglia (Streit et al. (1990), J Histochem Cytochem
38:1683-1686). Cells from hippocampus Cal were laser-captured, with
or without immunostaining. These cells were captured as they could
be identified without immunostaining and thus used as a control to
see the effects of immunostaining on the quality of the extracted
RNA. In addition to these cells, immunopositive microglia were also
laser-captured. The RNA from these laser-captured samples was
amplified using a T7-based aRNA amplification method, labelled with
Cy3 and hybridized onto cDNA microarrays.
[0092] Cluster analysis of the data from hippocampus Cal showed
that there was no portioning between the different experimental
conditions, as reflected in the dendrogram in FIG. 5. As shown in
the dendrogram, the one immunostained sample and one non-stained
sample were most closely related. Overall, the differences between
the samples were small. Cluster analysis of the microarray data did
not detect any differences between immunostained and non-stained
samples, showing that the RNA preservation maintained cellular RNA
content without bias.
[0093] As apparent from the foregoing, the inventive methods
robustly preserve RNA in a biological sample by using an RNA
preservative, providing advantageous methods for investigating mRNA
expression patterns within specific cell populations or
tissues.
[0094] While the above detailed description and preferred
embodiments have been provided to illustrate the invention and its
various features and advantages, it will be understood that
invention is defined not by the foregoing, but by the following
claims as properly construed under principles of patent law.
Sequence CWU 1
1
4 1 20 DNA Artificial PCR primer 1 cctccactgg catctatgag 20 2 20
DNA Artificial PCR primer 2 cctctatcgc cacattgctc 20 3 20 DNA
Artificial PCR primer 3 agatgaccta ggatgggagg 20 4 20 DNA
Artificial PCR primer 4 gtgtgcactg tgattcagac 20
* * * * *