U.S. patent application number 09/797158 was filed with the patent office on 2002-01-24 for isolation of rna from formalin-fixed paraffin-embedded tissue specimens.
Invention is credited to Danenberg, Kathleen, Danenberg, Peter V., Swenson, Steven.
Application Number | 20020009794 09/797158 |
Document ID | / |
Family ID | 23863395 |
Filed Date | 2002-01-24 |
United States Patent
Application |
20020009794 |
Kind Code |
A1 |
Danenberg, Kathleen ; et
al. |
January 24, 2002 |
Isolation of RNA from formalin-fixed paraffin-embedded tissue
specimens
Abstract
Methods are disclosed for rapid, reliable and simple isolation
of RNA from formalin-fixed paraffin-embedded tissue samples. RNA
purified in this manner can be used to monitor gene expression
levels. The tissue sample can be a tumor or other pathological
tissue.
Inventors: |
Danenberg, Kathleen;
(Altadena, CA) ; Danenberg, Peter V.; (Altadena,
CA) ; Swenson, Steven; (Arcadia, CA) |
Correspondence
Address: |
Rajiv Yadav
McCutchen, Doyle, Brown & Enersen, LLP
Three Embarcadero Center, 28th Floor
San Francisco
CA
94111
US
|
Family ID: |
23863395 |
Appl. No.: |
09/797158 |
Filed: |
March 1, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09797158 |
Mar 1, 2001 |
|
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09469338 |
Dec 20, 1999 |
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6248535 |
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Current U.S.
Class: |
435/270 ;
536/25.41 |
Current CPC
Class: |
C12N 15/1003 20130101;
C12Q 2600/158 20130101 |
Class at
Publication: |
435/270 ;
536/25.41 |
International
Class: |
C07H 021/02 |
Goverment Interests
[0002] The government has certain rights in this invention pursuant
to grant number R01 CA 71716 from the National Cancer Institute of
the National Institutes of Health.
Claims
What is claimed is:
1. A method for recovering RNA from a biological tissue sample
wherein the sample is not an aqueous sample of a bodily fluid,
comprising: heating the sample in a chaotropic solution comprising
an effective concentration of a guanidinium compound to a
temperature in the range of about 75 to about 100.degree. C. for a
time period of about 5 to about 120 minutes; and recovering said
RNA from said chaotropic solution.
2. The method of claim 1 further comprising rehydrating the sample
before heating.
3. The method of claim 2 further comprising homogenizing said
sample before beating.
4. The method of claim 3 wherein said RNA is recovered by
extraction from said chaotropic solution with a water insoluble
organic solvent.
5. The method of claim 4 wherein said water insoluble organic
solvent consists essentially of chloroform.
6. The method of claim 5 further comprising purifyng said RNA.
7. The method of claim 6 wherein said RNA is purified by ethanol
precipitation.
8. The method of claim 1 wherein said time period is from about 10
to about 60 minutes.
9. The method of claim 8 wherein said time period is from about 30
to about 60 minutes.
10. The method of claim 1 wherein said temperature is in the range
of about 85 to about 100.degree. C.
11. The method of claim 10 wherein said time period is from about
30 to about 60 minutes.
12. The method of claim 1 wherein said guanidinium compound is
guanidinium hydrochloride.
13. The method of claim 1 wherein said guanidinium compound is
guanidinium isothiocyanate.
14. The method of claim 13 wherein said guanidinium isothiocyanate
is present in a concentration of about 2 to about 5 M.
15. The method of claim 14 wherein said guanidinium isothiocyanate
is present in a concentration of about 4M.
16. The method of claim 13 wherein said chaotropic solution has a
pH of about 3-6.
17. The method of claim 16 wherein said chaotropic solution has a
pH of about 4.
18. The method of claim 1 wherein said chaotropic solution further
comprises a reducing agent.
19. The method of claim 18 wherein said reducing agent is
.beta.-mercaptoethanol.
20. The method of claim 18 wherein said reducing agent is
dithiothreitol.
21. The method of claim 1 wherein said RNA is used to determine the
level of expression of a gene.
Description
CROSS-REFERENCE TO RELATED APPLICATION DATA
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/469,338, filed Dec. 20, 1999.
FIELD OF THE INVENTION
[0003] This invention relates to the field of purification of RNA,
DNA and proteins from biological tissue samples.
BACKGROUND
[0004] The determination of gene expression levels in tissues is of
great importance for accurately diagnosing human disease and is
increasingly used to determine a patient's course of treatment.
Pharmacogenomic methods can identify patients likely to respond to
a particular drug and can lead the way to new therapeutic
approaches.
[0005] For example, thymidylate synthase (TS) is an integral enzyme
in DNA biosynthesis where it catalyzes the reductive methylation of
deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate
(dTMP) and provides the only route for de novo synthesis of
pyrimidine nucleotides within the cell (Johnston et al., 1995).
Thymidylate synthase is a target for chemotherapeutic drugs, most
commonly the antifolate agent 5-fluorouracil (5-FU). As the most
effective single agent for the treatment of colon, head and neck
and breast cancers, the primary action of 5-FU is to inhibit TS
activity, resulting in depletion of intracellular thymine levels
and subsequently leading to cell death. Considerable variation in
TS expression has been reported among clinical tumor specimens from
both primary tumors (Johnston et al., 1995; Lenz et al., 1995) and
metastases (Farrugia et al., 1997; Leichmann et al., 1997). In
colorectal cancer, for example, the ratio of TS expression in tumor
tissue relative to normal gastrointestinal mucosal tissue has
ranged from 2 to 10 (Ardalan and Zang, 1996).
[0006] Thymidylate synthase is also known to have clinical
importance in the development of tumor resistance, as demonstrated
by studies that have shown acute induction of TS protein and an
increase in TS enzyme levels in neoplastic cells after exposure to
5-FU (Spears et al. 1982; Swain et al. 1989). The ability of a
tumor to acutely overexpress TS in response to cytotoxic agents
such as 5-FU may play a role in the development of fluorouracil
resistance. Previous studies have shown that the levels of TS
protein directly correlate with the effectiveness of 5-FU therapy,
that there is a direct correlation between protein and RNA
expression (Jackman et al., 1985) and that TS expression is a
powerful prognostic marker in colorectal and breast cancer (Jackman
et al., 1985; Horikoshi et al., 1992).
[0007] In advanced metastatic disease, both high TS mRNA,
quantified by RT-PCR, and high TS protein expression, have been
shown to predict a poor response to fluoropyrimidinebased therapy
for colorectal (Johnston et al., 1995, Farrugia et al., 1997,
Leichman et al., 1997), gastric (Lenz et al., 1995, Alexander et
al., 1995), and head and neck (Johnston et al., 1997) cancers. A
considerable overlap between responders and non-responders was
often present in the low TS category, but patients with TS levels
above the median were predominantly nonresponders. The predictive
value of TS overexpression may be further enhanced if combined with
other molecular characteristics such as levels of dihydropyrimidine
dehydrogenase (DPD) and thymidine phosphorylase (TP) expression,
replication error positive (RER+) status (Kitchens and Berger
1997), and p53 status (Lenz et al., 1997). Studies to date that
have evaluated the expression of TS in human tumors suggest that
the ability to predict response and outcome based upon TS
expression in human tumors may provide the opportunity in the
future to select patients most likely to benefit from TS-directed
therapy.
[0008] Until now, quantitative tissue gene expression studies
including those of TS expression have been limited to reverse
transcriptase polymerase chain reaction (RT-PCR) amplification of
RNA from frozen tissue. However, most pathological samples are not
prepared as frozen tissues, but are routinely formalin-fixed and
paraffin-embedded (FFPE) to allow for histological analysis and for
archival storage. Gene expression levels can be monitored
semi-quantitatively and indirectly in such fixed and embedded
samples by using immunohistochemical staining to monitor protein
expression levels. Because paraffin-embedded samples are widely
available, rapid and reliable methods are needed for the isolation
of nucleic acids, particularly RNA, from such samples.
[0009] A number of techniques exist for the purification of RNA
from biological samples, but none are reliable for isolation of RNA
from FFPE samples. For example, Chomczynski (U.S. Pat. No.
5,346,994) describes a method for purifyng RNA from tissues based
on a liquid phase separation using phenol and guanidine
isothiocyanate. A biological sample is homogenized in an aqueous
solution of phenol and guanidine isothiocyanate and the homogenate
thereafter mixed with chloroform. Following centrifugation, the
homogenate separates into an organic phase, an interphase and an
aqueous phase. Proteins are sequestered in the organic phase, DNA
in the interphase, and RNA in the aqueous phase. RNA can be
precipitated from the aqueous phase. This method does not provide
for the reliable isolation of RNA from formalin-fixed
paraffin-embedded tissue samples.
[0010] Other known techniques for isolating RNA typically utilize
either guanidine salts or phenol extraction, as described for
example in Sambrook, J. et al., (1989) at pp. 7.3-7.24, and in
Ausubel, F. M. et al., (1994) at pp. 4.0.3-4.4.7. However, none of
the known methods provide reproducible quantitative results in the
isolation of RNA from paraffin-embedded tissue samples.
[0011] Techniques for the isolation of RNA from paraffin-embedded
tissues are particularly needed for the study of gene expression in
tumor tissues. Expression levels of certain receptors or enzymes
can indicate the likelihood of success of a particular
treatment.
[0012] Truly quantitative TS gene expression studies have been
limited to RT-PCR from frozen tissue, whereas semi-quantitative
monitoring of TS protein expression in archival pathological
material fixed onto glass slides has been available via
immunohistochemical staining. Because of limitations in isolating
RNA from archival pathological material, quantitative techniques
for measuring gene expression levels from such samples were
heretofore unavailable.
SUMMARY
[0013] One aspect of the present invention is to provide a reliable
method for the isolation of RNA, DNA or proteins from samples of
biological tissues. The invention also provides simple, efficient
and reproducible methods for the isolation of RNA, DNA or proteins
from tissue that has been embedded in paraffin.
[0014] The invention provides methods of purifying RNA from a
biological tissue sample by heating the sample for about 5 to about
120 minutes at a temperature of between about 50 and about
100.degree. C. in a solution of an effective concentration of a
chaotropic agent. In one embodiment, the chaotropic agent is a
guanidinium compound. RNA is then recovered from said solution. For
example, RNA recovery can be accomplished by chloroform
extraction.
[0015] In a method of the invention, RNA is isolated from an
archival pathological sample. In one embodiment, a
paraffin-embedded sample is first deparaffinized. An exemplary
deparaffinization method involves washing the paraffinized sample
with an organic solvent, preferably xylene. Deparaffinized samples
can be rehydrated with an aqueous solution of a lower alcohol.
Suitable lower alcohols include, methanol, ethanol, propanols, and
butanols. In one embodiment, deparaffinized samples are rehydrated
with successive washes with lower alcoholic solutions of decreasing
concentration. In another embodiment, the sample is simultaneously
deparaffinized and rehydrated.
[0016] The deparaffinized samples can be homogenized using
mechanical, sonic or other means of homogenization. In one
embodiment, the rehydrated samples are homogenized in a solution
comprising a chaotropic agent, such as guanidinium thiocyanate
(also sold as guanidinium isothiocyanate).
[0017] The homogenized samples are heated to a temperature in the
range of about 50 to about 100.degree. C. in a chaotropic solution,
comprising an effective amount of a chaotropic agent. In one
embodiment, the chaotropic agent is a guanidinium compound. A
preferred chaotropic agent is guanidinium thiocyanate.
[0018] RNA is then recovered from the solution by, for example,
phenol chloroform extraction, ion exchange chromatography or
size-exclusion chromatography.
[0019] RNA may then be further purified using the techniques of
extraction, electrophoresis, chromatography, precipitation or other
suitable techniques. RNA isolated by the methods of the invention
is suitable for a number of applications in molecular biology
including reverse transcription with random primers to provide cDNA
libraries.
[0020] Purified RNA can be used to determine the level of gene
expression in a formalin fixed paraffin-embedded tissue sample by
reverse transcription, polymerase chain reaction (RTPCR)
amplification. Using appropriate PCR primers the expression level
of any messenger RNA can be determined by the methods of the
invention. The quantitative RT-PCR technique allows for the
comparison of protein expression levels in paraffin-embedded (via
immunohistochemistry) with gene expression levels (using RT-PCR) in
the same sample.
[0021] The methods of the invention are applicable to a wide range
of tissue and tumor types and target genes and so can be used for
assessment of treatment and as a diagnostic tool in a range of
cancers including breast, head and neck, esophageal, colorectal,
and others. A particularly preferred gene for the methods of the
invention is the thymidylate synthase gene. The methods of the
invention achieved reproducible quantification of TS gene
expression in FFPE tissues, comparable to those derived from frozen
tissue.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1 shows level of .beta.-Actin and TS expression at
various heating times. These data show that without the heating
step, there is a minimal yield of RNA extracted from the
paraffin.
[0023] FIG. 2 shows the level of .beta.-actin expression in normal
(N) or tumorous (T) tissue from colorectal cancer patients as
determined by quantitative PCR from RNA extracted at 95.degree. C.
for zero to 40 minutes. These data suggest 30 min as an optimal
heating time.
[0024] FIG. 3 shows the effect of both temperature and time on the
yield of .beta.-actin RNA and on the isolation of DNA. These data
show that at longer heating times (between 60 and 120 min), RNA
undergoes degradation while there is an increase in contaminating
DNA capable of generating a DNA PCR signal. The bars represent
values of triplicate experiments done at the various times and
temperatures indicated.
[0025] FIG. 4 shows the effect of various heating solutions on the
yield of isolated RNA. These data show that the chaotrope in the
solution, in this case guanidinium isothiocyanate (GITC), is the
essential component of the RNA extraction solution, without which
the yield of extracted RNA is at least 10-fold lower.
[0026] FIG. 5 shows a comparison of relative TS gene expression
from paraffin-embedded (white bars) and frozen cell pellets (black
bars) from six cell lines. These data show that analysis of
paraffin-extracted RNA reliably reflects gene expression values in
fresh-frozen tissue.
[0027] FIG. 6 shows a comparison of TS gene expression levels in
normal or tumorous colon and tumorous esophageal tissues that were
either formalin-fixed and paraffin-embedded or frozen.
[0028] FIG. 7 shows TS/.beta.-actin ratios determined in paraffin
sections from patients whose response to 5-FU/LV was previously
linked to TS gene expression.
[0029] FIG. 8 shows the expression levels of four malignancy marker
genes (TS; thymidine phosphorylase (TP); cyclooxygenase-2 (COX-2);
and vascular endothelial growth factor (VEGF)) in FFPE samples of a
primary colon cancer and a liver metastasis that recurred a year
later in the same patient. These data show that, as might be
expected, three of the four malignancy markers are elevated in the
metastatic tumor tissue.
DETAILED DESCRIPTION
[0030] The methods of the instant invention involve purification of
RNA from biological samples. In one embodiment, samples are
paraffin-embedded tissue samples and the method involves
deparaffinization of embedded samples, homogenization of the
deparaffinized tissue and heating of the homogenized tissue at a
temperature in the range of about 50 to about 100.degree. C. for a
time period of between about 5 minutes to about 120 minutes in a
chaotropic solution containing an effective amount of a guanidinium
compound. This heating step increases the amount of cDNA that are
amplified from the specimen by up to 1000-fold over samples that
are not heated.
[0031] While frozen tumor tissue is not widely available, paraffin
blocks are routinely prepared from every type of tumor after
surgery, allowing large-scale retrospective investigations of TS
expression and chemotherapy response to be performed.
[0032] Moreover, the technique can be applied to any of a wide
range of tumor types and to an unlimited range of target genes.
This has implications for the future preparation of individual
tumor "gene expression profiles" whereby expression levels could be
determined in individual patient samples for a range of genes that
are known to influence clinical outcome and response to various
chemotherapeutic agents. Automated real-time PCR from FFPE sample
allows for the targeting of treatment to individual tumors.
Tissue Samples
[0033] RNA can be isolated from any biological sample using the
methods of the invention. Biological samples are often fixed with a
fixative. Aldehyde fixatives such as formalin (formaldehyde) and
glutaraldehyde are typically used. Tissue samples fixed using other
fixation techniques such as alcohol immersion (Battifora and
Kopinski, J. Histochem. Cytochem. (1986) 34:1095) are also
suitable. The samples used are also embedded in paraffin. RNA can
be isolated any paraffin-embedded biological tissue sample by the
methods of the invention. In one embodiment, the samples are both
formalin-fixed and paraffin-embedded.
Deparaffinization of Samples
[0034] Deparaffinization removes the bulk of paraffin from the
paraffin-embedded sample. A number of techniques for
deparaffinization are known and any suitable technique can be used
with the present invention. The preferred method of the invention
utilizes washing with an organic solvent to dissolve the paraffin.
Such solvents are able to remove paraffin effectively from the
tissue sample without adversely affecting RNA isolation. Suitable
solvents can be chosen from solvents such as benzene, toluene,
ethylbenzene, xylenes, and mixtures thereof. A xylene is the
preferred solvent for use in the methods of the invention. Solvents
alone or in combination in the methods of the invention are
preferably of high purity, usually greater than 99%.
[0035] Paraffin is typically removed by washing with an organic
solvent, with vigorous mixing followed by centrifugation. Samples
are centrifuged at a speed sufficient to cause the tissue to pellet
in the tube, usually at about 10,000 to about 20,000.times.g. After
centrifugation, the organic solvent supernatant is discarded. One
of skill in the art of histology will recognize that the volume of
organic solvent used and the number of washes necessary will depend
on the size of the sample and the amount of paraffin to be removed.
The more paraffin to be removed, the more washes that will be
necessary. Typically, a sample will be washed between 1 and about
10 times, and preferably, between about two and about four times. A
typical volume of organic solvent is about 500 .mu.L for a 10 .mu.m
tissue specimen.
[0036] Other methods for deparaffinization known to one of skill in
the art may also be used in the method of the invention. Such
methods include direct melting (Baneijee et al., 1995).
[0037] Samples are preferably rehydrated after deparaffinization.
The preferred method for rehydration is step-wise washing with
aqueous lower alcoholic solutions of decreasing concentration.
Ethanol is a preferred lower alcohol for rehydration. Other
alcohols may also be suitable for use with the invention including
methanol, isopropanol and other similar alcohols in the C1-C5
range. The sample is alternatively vigorously mixed with alcoholic
solutions and centrifuged. In a preferred embodiment, the
concentration range of alcohol is decreased stepwise from about
100% to about 70% in water over about three to five incremental
steps, where the change in solution concentration at each step is
usually less than about 10% (i.e., the sequence: 100%, 95%, 90%,
80%, 70%). In another embodiment, deparaffinization and rehydration
are carried out simultaneously using a reagent such as EZ-DEWAX
(BioGenex, San Ramon, Calif.).
Homogenization
[0038] Deparaffmized, rehydrated samples can be homogenized by any
standard mechanical, sonic or other suitable technique. Tissue
homogenization is preferably carried out with a mechanical tissue
homogenizers according to standard procedures. A number of
commercially available homogenizers are suitable for use with the
invention including: Ultra-Turrax homogenizer (IKA-Works, Inc.,
Wilmington, N.C.); Tissumizer (Tekmar-Dohrmann, Cincinnati, Ohio);
and Polytron (Brinkmann, Westbury, N.Y.).
[0039] In one embodiment, the sample is homogenized in the presence
of a chaotropic agent. Chaotropic agents are chosen such that at an
effective concentration RNA is purified from a paraffin-embedded
sample in an amount of greater than about 10 fold that isolated in
the absence of a chaotropic agent. Chaotropic agents include:
guanidinium compounds, urea, formarnide, potassium iodiode,
potassium thiocyantate and similar compounds. The preferred
chaotropic agent for the methods of the invention is a guanidinium
compound, such as guanidinium isothiocyanate (also sold as
guanidinium thiocyanate) and guanidinium hydrochloride. Many
anionic counterions are useful, and one of skill in the art can
prepare many guanidinium salts with such appropriate anions. The
guanidinium solution used in the invention generally has a
concentration in the range of about 1 to about 5M with a preferred
value of about 4M. If RNA is already in solution, the guanidinium
solution may be of higher concentration such that the final
concentration achieved in the sample is in the range of about 1 to
about 5M. The guanidinium solution also is preferably buffered to a
pH of about 3 to about 6, more preferably about 4, with a suitable
biochemical buffer such as Tris-Cl. The chaotropic solution may
also contain reducing agents, such as dithiothreitol (DTT) and
.beta.-mercaptoethanol (BME). The chaotropic solution may also
contain RNAse inhibitors.
Heating
[0040] Samples are heated in the chaotropic solution at a
temperature of about 60.degree. C. to about 100.degree. C. for
about 5 minutes to about 2 hours. Alternatively, samples are heated
in the chaotropic solution at a temperature of about 50.degree. C.
to about 100.degree. C. for about 5 minutes to about 2 hours.
Heating times are typically chosen such that the amount of RNA
purified is at least about 100-fold higher than for unheated
samples, and more preferably about 1000-fold higher. In a preferred
embodiment, the sample is heated for about 20 minutes at a
temperature of from about 75 to about 100.degree. C. More
preferably, the sample is heated for 30 to 60 minutes at about
95.degree. C.
RNA Recovery
[0041] RNA can be recovered from the chaotropic solution after
heating by any suitable technique that results in isolation of the
RNA from at least one component of the chaotropic solution. RNA can
be recovered from the chaotropic solution by extraction with an
organic solvent, chloroform extraction, phenol-chloroform
extraction, precipitation with ethanol, isopropanol or any other
lower alcohol, by chromatography including ion exchange
chromatography, size exclusion chromatography, silica gel
chromatography and reversed phase chromatography, or by
electrophoretic methods, including polyacrylamide gel
electrophoresis and agarose gel electrophoresis, as will be
apparent to one of skill in the art. RNA is preferably recovered
from the chaotropic solution using phenol chloroform
extraction.
[0042] Following RNA recovery, the RNA may optionally by further
purified. Further purification results in RNA that is substantially
free from contaminating DNA or proteins. Further purification may
be accomplished by any of the aforementioned techniques for RNA
recovery. RNA is preferably purified by precipitation using a lower
alcohol, especially with ethanol or with isopropanol. Precipitation
is preferably carried out in the presence of a carrier such as
glycogen that facilitates precipitation.
DNA and Protein Recovery
[0043] The methods of the invention can also be used to purify DNA
or protein from the tissue sample. After mixing a sample with an
organic solvent, such as chloroform, and following centrifugation,
the solution has three phases, a lower organic phase, an
interphase, and an upper aqueous phase. With an appropriate
chaotropic agent, particularly with a guanidinium compound, the
biological components of the sample will segregate into the three
phases. The upper aqueous phase will contain RNA, while the
interphase will contain DNA and the organic phase will contain
proteins. One of skill in the art will recognize that the methods
of the invention are suitable for recovering both the DNA and
protein phases as well as that containing the RNA. DNA recovery is
enhanced by the methods of the invention.
Purified RNA
[0044] RNA purified by the methods of the invention is suitable for
a variety of purposes and molecular biology procedures including,
but not limited to: reverse transcription to cDNA; producing
radioactively, fluorescently or otherwise labeled cDNA for analysis
on gene chips, oligonucleotide microarrays and the like;
electrophoresis by acrylamide or agarose gel electrophoresis;
purification by chromatography (e.g. ion exchange, silica gel,
reversed phase, or size exclusion chromatography); hybridization
with nucleic acid probes; and fragmentation by mechanical, sonic or
other means.
EXAMPLES
Materials and Methods
[0045] These materials and methods are common to the following
examples.
[0046] Sample Preparation. The characteristics of the human cell
lines SK1, H157, A431, HT29, HCC298 and HH30 have been described
previously. Cells were removed from their monolayer by
trypsinization and pelleted by centrifugation at 3000 rpm for 5
minutes. Cell pellets were either frozen at -70.degree. C. or fixed
in formalin for 24h, then embedded in paraffin.
[0047] Representative sections of tumor tissue were obtained at the
time of surgery, fixed in formalin and embedded in paraffin by
procedures common to most clinical pathology laboratories.
Cross-sections of tissue (5 .mu.m) were cut using a microtome.
[0048] RNA Isolation. RNA was isolated from paraffin embedded
tissue as follows. A single 5 .mu.m section of paraffmized tissue
was placed in an Eppendorf tube and deparaffinized by two 15 minute
washes with xylene. The tissue was rehydrated by successive 15
minute washes with graded alcohols (100%, 95%, 80% and 70%). The
resulting pellet was suspended in 4M guanidine isothiocyanate with
0.5% sarcosine and 20 mM dithiothreitol (DTT). The suspension was
homogenized and then heated to from about 50 to about 95.degree. C.
for 0 to 60 minutes; a zero heating time-point, was included as a
control for each sample. Sodium acetate (pH 4.0) was added to 0.2 M
and the solution was extracted with phenol/chloroform and
precipitated with isopropanol and 10 mg glycogen. After
centrifugation (13000 rpm, 4.degree. C., 15 min) the RNA pellet was
washed twice with 1 mL of 75% ethanol then resuspended in
RNase-free water.
[0049] Reverse transcription (RT). After heating, total RNA was
converted to cDNA using random hexamers. RT conditions were as have
been previously described for frozen tissue (Horikoshi et al.,
1992). Controls omitting the reverse transcriptase (No-RT) were
prepared for each sample.
[0050] Real-Time PCR quantification of TS and .beta.-actin gene
expression using the Perkin Elmer Cetus 7700 PCR Machine (Taqman).
The quantitation of mRNA levels was carried out using real-time PCR
based on a fluorescence detection method as described previously
(Heid et al., 1996; Eads et al., 1999). cDNA was prepared as
described above. The cDNA of interest and the reference cDNA were
amplified separately using a probe with a 5'-fluorescent reporter
dye (6FAM) and a 3'-quencher dye (TAMRA). The 5'-exonuclease
activity of TAQ polymerase cleaves the probe and releases the
reporter molecule, the fluorescence of which is detected by the ABI
Prism Sequence Detection System (Taqman). After crossing a
fluorescence detection threshold, the PCR amplification results in
a fluorescent signal proportional to the amount of PCR product
generated. Initial template concentration was determined from the
cycle number at which the fluorescent signal crossed a threshold in
the exponential phase of the PCR reaction. Relative gene expression
was determined based on the threshold cycles of the gene of
interest and the reference gene. Use of a reference gene avoids the
need to quantitate the RNA directly, which could be a major source
of error.
[0051] The primer and probe sequences were as follows: TS: SEQ ID
NO: 1: GGC CTC GGT GTG CCT TT; SEQ ID NO: 2: AAC ATC GCC AGC TAC
GCC CTG C; SEQ ED NO: 3: GAT GTG CGC AAT CAT GTA CGT. .beta.-actin:
SEQ ID NO: 4: TGA GCG CGG CTA CAG CTT; SEQ ID NO: 5: ACC ACC ACG
GCC GAG CGG; SEQ ID NO: 6: TCC TTA ATG TCA CGC ACG ATT T. For the
real-time PCR experiments, as discussed above, the reporter
oligonucleotide (SEQ ID NOS: 2 and 5) were 5' labelled with 6FAM
and were 3' labelled with TAMRA.
[0052] For each PCR, a "No Reverse Transcriptase" (NRT or No-RT)
control was included. The purpose of this reaction was to correct
for any background amplification, derived from residual genomic DNA
contamination. Hence, each overall value for TS and .beta.-actin is
calculated as the RT value minus the NRT value (RT-NRT).
[0053] Statistical Analysis. Non-parametric comparison of means
test were performed to determine if differences in TS levels
between frozen tissue and FFPE samples of the same tumor were
significant or non-significant.
EXAMPLE 1
General RNA Isolation Procedure
[0054] RNA was extracted from paraffin-embedded tissue by the
following general procedure.
[0055] A. Deparaffinization and hydration of sections:
[0056] (1) A portion of an approximately 10 .mu.M section is placed
in a 1.5 mL plastic centrifuge tube.
[0057] (2) 600 .mu.L of xylene are added and the mixture is shaken
vigorously for about 10 minutes at room temperature (roughly 20 to
25.degree. C.).
[0058] (3) The sample is centrifuged for about 7 minutes at room
temperature at the maximum speed of the bench top centrifuge (about
10-20,000.times.g).
[0059] (4) Steps 2 and 3 are repeated until the majority of
paraffin has been dissolved. Two or more times are normally
required depending on the amount of paraffm included in the
original sample portion.
[0060] (5) The xylene solution is removed by vigorously shaking
with a lower alcohol, preferably with 100% ethanol (about 600
.mu.L) for about 3 minutes.
[0061] (6) The tube is centrifuged for about 7 minutes as in step
(3). The supernatant is decanted and discarded. The pellet becomes
white.
[0062] (7) Steps 5 and 6 are repeated with successively more dilute
ethanol solutions: first with about 95% ethanol, then with about
80% and finally with about 70% ethanol.
[0063] (8) The sample is centrifuged for 7 minutes at room
temperature as in step (3). The supernatant is discarded and the
pellet is allowed to dry at room temperature for about 5
minutes.
[0064] B. RNA Isolation with Phenol-Chloroform
[0065] (1) 400 .mu.L guanidine isothiocyanate solution including
0.5% sarcosine and 8 .mu.L 1M dithiothreitol is added.
[0066] (2) The sample is then homogenized with a tissue homogenizer
(Ultra-Turrax, IKA-Works, Inc., Wilmington, N.C.) for about 2 to 3
minutes while gradually increasing the speed from low speed (speed
1) to high speed (speed 5).
[0067] (3) The sample is then heated at about 95.degree. C. for
about 5-20 minutes. It is preferable to pierce the cap of the tube
containing the sample before heating with a fine gauge needle.
Alternatively, the cap may be affixed with a plastic clamp or with
laboratory film.
[0068] (4) The sample is then extracted with 50 .mu.L 2M sodium
acetate at pH 4.0 and 600 .mu.L of phenol/chloroform/isoamyl
alcohol (10:1.93:0.036), prepared fresh by mixing 18 mL phenol with
3.6 mL of a 1:49 isoamyl alcohol:chloroforn solution. The solution
is shaken vigorously for about 10 seconds then cooled on ice for
about 15 minutes.
[0069] (5) The solution is centrifuged for about 7 minutes at
maximum speed. The upper (aqueous) phase is transferred to a new
tube.
[0070] (6) The RNA is precipitated with about 10 .mu.L glycogen and
with 400 .mu.L isopropanol for 30 minutes at -20.degree. C.
[0071] (7) The RNA is pelleted by centrifugation for about 7
minutes in a benchtop centrifuge at maximum speed; the supernatant
is decanted and discarded; and the pellet washed with approximately
500 .mu.L of about 70 to 75% ethanol.
[0072] (8) The sample is centrifuged again for 7 minutes at maximum
speed. The supernatant is decanted and the pellet air dried. The
pellet is then dissolved in an appropriate buffer for further
experiments (e.g. 50 .mu.L 5mM Tris chloride, pH 8.0).
EXAMPLE 2
Heating Time
[0073] This example illustrates the effect of time of heating on
the yield of RNA.
[0074] As illustrated in FIG. 1, heating of the chaotropic solution
at 95.degree. C. prior to precipitation and reverse transcription
significantly increased the efficiency of detection of TS and
.beta.-actin targets. When no heating step was included, neither TS
nor .beta.-actin could be detected (0 min. time points). After 20
minutes at 95.degree. C. both transcripts were detectable; a
further increase of heating time to 60 minutes resulted in a 3-fold
increase in sensitivity of detection for TS and 4.5-fold increase
for .beta.-actin. (NRT=No Reverse Transcriptase control,
RT-NRT=overall relative gene expression level, i.e. Reverse
Transcriptase minus No Reverse Transcriptase).
[0075] FIG. 2 illustrates the amount of RNA expression of the
.beta.-actin gene in normal (N) and tumorous (T) tissue. The
samples were heated at 95.degree. C. for periods ranging from zero
to 40 minutes. A preferred heating time of about 30 minutes is
observed for most samples.
[0076] FIG. 3 shows that at heating times longer than about 60 min,
the amount of RNA extracted starts to decrease, suggesting thermal
degradation of the RNA, whereas the amount of DNA extracted starts
to increase. This is undesirable because the presence of DNA can
give a spurious PCR signal in some cases.
EXAMPLE 3
Heating Solutions
[0077] This example illustrates that heating the RNA solution in
the presence of a chaotropic agent is important for obtaining high
yields of RNA. This was an RT-PCR experiment using detection of
.beta.-actin gene expression as a measure of relative amounts of
RNA isolated in various solutions.
[0078] Clinical specimens of esophageal cancer FFPE tissue samples
were treated according to the methods described above, with the
exception that the initial pellet obtained after deparaffinization
was dissolved or suspended in either 4M guanidinium isothiocyanate
(GITC), 4M guanidinium isothiocyanate+100 .mu.M
.beta.-mercaptoethanol (GITC+BME), 4M guanidinium isothiocyanate+20
.mu.M dithiothreitol (GITC+DTT) or in Tris-Cl buffer (10 mM pH 7.5)
or Tris-Cl buffer+20 pM DTT (Tris/Cl+DTT). The samples were then
heated to 95.degree. C. for 30 minutes or not heated (0 min,
95.degree. C.). The Tris/Cl samples were then treated with 4M
guanidinium isothiocyanate. RNA levels were determined by RT-PCR
and real time PCR detection of .beta.-Actin. As shown in FIG. 4,
the presence of the chaotropic agent guanidinium isothiocyanate
when heating was important for high yield recovery of RNA. The
presence of a reducing agent, such as DTT or BME, is not essential
for high yield recovery of RNA. The 4M guanidinium isothiocyanate
solution contains 50 mM Tris-HCl (pH 7.5), 25 mM EDTA and 0.5%
Sarcosine.
EXAMPLE 4
Comparison of Gene Expression Values Determined in FFPE and Frozen
Tissue from the Same Sources
[0079] This example shows that the methods of the present invention
provide values for gene expressions from formalin-fixed
paraffin-embedded samples equivalent to those obtained from frozen
tissue.
[0080] Samples from six cell lines were FFPE-treated and TS
quantitation performed using the methods of the invention
(including heating at 95.degree. C. for 30 minutes). The resulting
relative TS values (FIG. 5) were compared with those obtained from
frozen cell pellets using known methods. Relative TS expression
levels were 3.0-19.5 (mean=8.5) in frozen cells versus 3.0-25.0
(mean=9.0) in FFPE samples. Statistical analysis of the difference
between the two means revealed a p value of 0.726, indicating that
there is no significant difference in the TS values obtained from
frozen cell pellets using the original RT-PCR methods and those
obtained from FFPE cell pellets using the methods of the
invention.
[0081] RNA expression levels in samples of tumorous tissues and of
normal (non-tumorous) tissues also were equivalent regardless of
whether the samples were formalin-fixed and paraffin-embedded or
frozen. Five normal and 6 tumor colon tissues and 4 esophageal
tumor tissues, were compared for relative TS gene expression in
matching paraffin and frozen tissue (FT) as above. Results are
illustrated in FIG. 6. No significant difference was found between
the levels of TS found in frozen tissue samples and the TS values
found in FFPE samples of the same tissue. This was true for both
colon and esophageal tissue types (mean FT samples colon=3.46, mean
FFPE samples colon=3.06, p=0.395; mean FT samples esophagus=13.9,
mean FFPE samples esophagus=15.93, p=0.21).
EXAMPLE 5
Comparison of TS Levels in Responsive and Non-Responsive Tumor
Tissues
[0082] Correlation of TS levels in frozen tissue and matching FFPE
samples with response to 5-FU/Leucovorin (LV) in stage IV colon
cancer. Previous reports based on RT-PCR data derived from frozen
tissue found that high levels of TS in tumors (relative gene
expression .gtoreq.4.0) were indicative of a poor response to TS
treatment. Responsive tumors could be characterized as expressing
lower levels of TS. TS/.beta.-actin ratios were determined in
paraffin sections from 17 patients whose response to 5-FU/LV had
previously been linked to TS gene expression via analysis of frozen
tissue samples (FIG. 7). Of the 17, 6 were known to be responsive
to TS and 11 were known to have been poor responders to TS
treatment. It was found that the TS results with matching paraffin
tissue would also have predicted response to this therapy (mean
responders FT=2.87, mean responders FFPE=2.37, p=0.641: mean
nonresponders FT=7.66, mean non-responders FFPE=7.84 p=0.537).
There was no significant difference between the TS levels derived
from frozen tissue and those derived from matching FFPE
tissues.
EXAMPLE 6
TS Gene Expression Levels in Primary Colon Cancer and a Liver
Metastasis
[0083] This example shows an analysis of TS, and other gene
expression, in a primary colon tumor and in a recurrent liver
metastasis from the same patient.
[0084] FIG. 8 shows the expression levels of four genes: TS; TP;
cyclooxygenase-2 (COX-2); and vascular endothelial growth factor
(VEGF) in FFPE samples of a primary colon cancer and a liver
metastasis (met) from the same patient which recurred a year later.
The findings suggest that, while the primary tumor was sensitive to
5-FU therapy (TS=2.32), the metastasis will be refractory (TS met
11.58). COX-2 and VEGF expression levels correlate with the
published indications that they are increased in expression in
aggressive disease, and coregulated. (Cox-2 primary=1.35; COX-2
met=5.4; VEGF primary=5.02; VEGF met=14.4.) RNA was isolated as
described from a 5 .mu.M FFPE section of the primary colon cancer
and from an FFPE section of the liver metastasis. Relative TS gene
expression in the responsive primary tumor was 2.32 compared to
11.58 in the metastastic disease (FIG. 8). This 5-fold increase in
TS expression, as determined by the RT-PCR methods reported here,
indicates that the secondary disease will not respond to 5-FU and
suggests an alternative therapy such as CPT-11 may be
appropriate.
[0085] All references cited herein are hereby incorporated by
reference in their entirety.
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