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From the Departments of Laboratory Medicine
*
and Surgery,
University of California San Francisco Cancer Center, San Francisco, California; P.E. Biosystems,
Foster City, California; and the School of Dentistry,
Chonnam National University, Kwangju, South Korea
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although many institutions are now maintaining frozen tumor banks, which should facilitate gene expression analyses in the future, few of these now have sufficient clinical follow-up to be useful for retrospective studies correlating gene expression with clinical outcome. On the other hand, there is a vast supply of formalin-fixed, paraffin-embedded tumor tissues for which response to treatment and clinical outcome is already known. Although these archived tissues can be used for in situ techniques to show localization of gene expression,4, 5 the RNA is too degraded for classical, quantitative analysis methods such as Northern blots. Reverse transcription-polymerase chain reaction (RT-PCR) has been used extensively to detect expression of genes in cultured cells and in fresh or frozen tissues, and recent technological advances now allow rapid and accurate quantitative RT-PCR analyses.6, 7 Furthermore, the ability of RT-PCR to assay very small fragments of mRNAs makes this technique amenable to studies where the RNA is moderately or even highly degraded, as in the case of RNA from archived tissues.8, 9
Although it has previously been demonstrated that RNA from formalin-fixed tissues can be used for RT-PCR10, 11, 12, 13, 14, 15 and that the RT-PCR steps can be done in a quantitative manner,16, 17 it has not been shown that RNA levels measured in archival tissues accurately reflect expression in the tissue before fixation. Several factors may influence relative RNA representation in fixed versus fresh tissues and some of these, such as time in fixative and type of fixative, have already been addressed by others.8, 9, 18, 19 Other important factors that have not been addressed previously include RNA half-life, time from surgical excision to fixation (pre-fixation time), and differential effect of fixation on RNA populations and the subsequent ability to extract and quantitate all RNA species equally.
The purpose of this study was to determine the effect of these variables on quantitation of gene expression as measured using TaqMan RT-PCR methodology7 and thus determine the feasibility of gene expression studies on archival material.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After fixation, samples were dehydrated, incubated in xylene, incubated in paraffin, and embedded in paraffin using a Leica Paraffin-Embedder (Leica Microsystems Inc., Deerfield, IL).
RNA Extraction
RNA isolation from paraffin tissue sections was based on the
methods first described by Fisher.20
Paraffin-embedded
tissue samples were cut into 5 x 5 µm to 50 x 5
µm sections, depending on the size of the embedded tissue
sample, and placed in RNase-free, 2.0-ml Eppendorf tubes. Sections were
deparaffinized by incubation in 1.8 ml of xylene or Americlear
(Stephens Scientific, Riverdale, NJ) at 37°C for 20 minutes.
The samples were then centrifuged, the supernatant was removed, and
fresh xylene was added for a second incubation. After deparaffinization
and centrifugation, sections were washed with 0.5 ml ethanol, air-dried
for several minutes, and resuspended in 80 µl of 60 mg/ml (20 U/mg)
Proteinase K (Gibco BRL, Gaithersburg, MD) plus 720 µl of a digestion
buffer with the following final concentrations: 1 mol/L guanidinium
thiocyanate, 25 mmol/L 2-mercaptoethanol, 0.5% Sarcosyl 20
(N-lauroylsarcosine), 20 mmol/L Tris-HCl, pH 7.5.10
Samples were vortexed and incubated overnight at 55°C. A second
80-µl aliquot of 60 mg/ml Proteinase K was then added, followed by
vortexing and a second overnight incubation at 55°C. On the third
day, a final aliquot of Proteinase K was added and the samples were
again vortexed, followed by overnight incubation at 55°C. RNA was
obtained by extraction with an equal volume of 70% phenol (pH
4.3):30% chloroform at room temperature. Samples were centrifuged for
5 minutes at 14,000 rpm and the aqueous phase was transferred to new,
RNase-free Eppendorf tubes. The RNA was precipitated by addition of an
equal volume of isopropanol, along with 2 µg glycogen, at -20°C
for 30 minutes. The samples were then centrifuged for 30 minutes at
14,000 rpm, the RNA pellets were washed in 70% EtOH, air-dried for
several minutes on the bench, and then resuspended in 20 µl diethyl
pyrocarbonate (DEPC)-treated H2O. Trizol
reagent (500 µl) was then added to each RNA sample and purification
was performed according to the manufacturer’s instructions with one
exception: following the first Trizol treatment, the aqueous phase was
not precipitated but was instead subjected to a second 500-µl Trizol
extraction. The aqueous phase from this step was used for RNA
precipitation with 500 µl of isopropanol and the RNA was resuspended
in 20 µl deionized formamide for storage at -20°C. All RNAs were
quantitated by spectrophotometer and OD 260/280 nm ratios >1.8 were
obtained for all samples, indicating high purity. All solutions,
including digestion buffer and ethanol/water solutions, were made using
DEPC-treated water.
RNA Isolation from Fresh Tissue/Cell Lines
RNA was isolated from fresh tissues using Trizol reagent according
to the manufacturer’s instructions, with the exception that a second
Trizol extraction was performed as described above. RNA was resuspended
in 20 µl of deionized formamide for storage before a final
purification step with RNeasy columns (Qiagen, Valencia, CA) according
to the manufacturer’s protocol. RNA was quantitated as described
above.
Reverse Transcription
The optimal reverse transcription (RT) was carried out in 100-µl
volumes consisting of 1x PCR buffer II (PE Biosystems, Foster City,
CA), 250 units of Moloney-murine leukemia virus (M-MLV) reverse
transcriptase (Life Technologies), 40 units of RNase inhibitor (Roche
Molecular Biochemicals, Indianapolis, IN), 7.5 mmol/L MgCl, 1
mmol/L each dNTP (Roche Molecular Biochemicals), 5 µmol/L random
hexamers (Life Technologies), and 75 to 300 ng total RNA. Reactions
were incubated at 25°C for 10 minutes, 48°C for 30 minutes, and
95°C for 5 minutes in a Perkin-Elmer GeneAmp PCR System 9700. "No
RT " controls were carried out in all cases using the same RT
reaction mix but substituting DEPC-H2O for M-MLV
reverse transcriptase. For all quantitative analyses 3 RTs were carried
out for each RNA sample. The RNA amounts used in each RT were 300, 150,
and 75 ng per 100-µl RT reaction. All "no RT" controls were
carried out with 300 ng of RNA.
Real Time Quantitative RT-PCR
Relative abundance of each mRNA species was assessed using the 5'
fluorogenic nuclease assay to perform real time quantitative
PCR.7, 21, 22, 23
The basis for this system is to continuously
measure PCR product accumulation using a dual-labeled fluorogenic
oligonucleotide probe, called a TaqMan probe. This probe is composed of
a short (
20–25 bases) oligodeoxynucleotide labeled with two
different fluorescent dyes. The oligonucleotide probe sequence is
homologous to an internal target sequence present in the PCR amplicon.
When the probe is intact, energy transfer occurs between the two
fluorophors and emission from the reporter is quenched by the quencher.
During the extension phase of PCR, the probe is cleaved by 5' nuclease
activity of Taq polymerase, thereby releasing the reporter
from the oligonucleotide quencher and producing an increase in reporter
emission intensity. The ABI Prism 7700 detects and plots this increase
in fluorescence versus PCR cycle number to produce a
continuous measure of PCR amplification. To provide precise
quantification of initial target in each PCR reaction, the
amplification plot is examined at a point during the early log phase of
product accumulation. This is accomplished by assigning a fluorescence
threshold above background and determining the time point at which each
sample’s amplification plot reaches the threshold (defined as the
threshold cycle number or CT). Differences in
threshold cycle number are used to quantify the relative amount of PCR
target contained within each tube as described
previously.24, 25
The following genes were studied: glyceraldehyde phosphate
dehydrogenase (GAPDH),26
ß-glucuronidase
(ß-Gus),27
c-myc,28
erbB-2,29
ß-actin,30
ZNF217,25
vascular endothelial growth factor
(VEGF),31
and VEGF receptors Flt32
and
KDR.33
PCR primers and fluorogenic probe (TaqMan) were
designed using Primer Express software (PE Biosystems), and the
sequences are given in Table 1
. Wherever possible, TaqMan primers and probes were designed to span
introns in the genomic DNA, thereby minimizing signal from
contaminating genomic DNA. All probes were obtained from either PE
Biosystems or Synthetic Genetics (San Diego, CA). Primers were obtained
from PE Biosystems or Life Technologies. Quantitative RT-PCR was
performed in duplicate in 50-µl reaction volumes consisting of 1x
PCR buffer A (PE Biosystems), 5.5 mmol/L MgCl2,
0.025 U/µl AmpliTaq Gold (PE Biosystems), and 5 µl of the
appropriate RT reaction. The final primer and probe concentrations used
for each gene are shown in Table 2
. Two-step PCR cycling was carried out as follows: 95°C 12
minutes x 1 cycle, 95°C 15 seconds, 60°C 1 minute x 40
cycles. At the end of the PCR, baseline and threshold values were set
in the ABI 7700 Prism software and the calculated
CT values were exported to Microsoft Excel for
analysis.
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For each RNA sample (including the fresh tissue sample), a difference
in CT values (
CT) was
calculated for each mRNA by taking the mean CT of
duplicate tubes and subtracting the mean CT of
the duplicate tubes for the reference RNA (ß-Gus) measured on an
aliquot from the same RT reaction.
CT = CT(test gene) - CT(ß-Gus)
The CT for the fresh tissue sample was then
subtracted from the CT for the test sample to
generate a CT.
CT = CT(test
RNA) - CT(fresh tissue RNA)
Since all RNAs were reverse transcribed at 3 different RNA
concentrations, 3 values of CT were
calculated for each gene on each RNA sample:
CT(300 ng),
CT(150 ng), and
CT(75 ng). The mean of these
CT measurements was then used to calculate
expression of the test gene relative to the reference gene and
normalized to the fresh tissue sample as follows:
Relative Expression = 2-CT
This calculation assumes that all PCR reactions are working with 100% efficiency. All PCR efficiencies were measured as previously described24 and were found to be >95%; therefore, this assumption introduces minimal error into the calculations.
Error bars were plotted as 80% confidence limits based on the average
SD of all triplicate CT measurements within
the experiment.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and RNA length as determined by denaturing gel electrophoresis
(Figure 1A)
indicated that a 3-day proteinase K incubation gave
increased yield and RNA length. However, the OD260/280 ratios obtained
with this procedure were relatively low (1.3–1.6), indicating
impurities in the RNA. Furthermore, RT reactions were inconsistent,
especially at higher RNA inputs, suggesting that these impurities were
inhibiting the RT reaction in an unpredictable manner, thus making
quantitative RT-PCR difficult. We believe that this is due to
proteinase K carried over from the RNA extraction procedure
despite heat inactivation. We have previously observed that small
amounts of proteinase K activity can survive phenol extraction and heat
inactivation, and will subsequently inactivate RT-PCR enzymes (Ruff DW,
unpublished results). We found that two sequential Trizol extractions
were sufficient to increase the OD260/280 ratios to >1.6, and made the
subsequent RT steps much more consistent and quantitative. This extra
purification step also has the added advantage of removing most of the
genomic DNA from the RNA sample, thus making the "no RT" controls
consistently negative.
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Effect of Amplicon Size on Quantitation
To test the effect of different PCR amplicon sizes on mRNA
quantitation we assayed 99- to 291-bp fragments of the ß-actin
mRNA. In the first experiment we looked at absolute
CT values obtained for actin of 99, 115, 131,
175, 205, and 291 bp on high quality RNA from fresh liver tissue RNA
versus the immediate fix and 12-hour pre-fixation RNAs
(Figure 2)
. It can be seen that the relative abundance of quantifiable ß-actin
fragments is lower (higher CT) in the fixed
tissues than in the fresh tissue RNA and that this difference in
abundance is greatest at the larger fragment sizes.
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. It can be seen that for the fixed tissues the abundance of the 291-bp
actin fragment was approximately 90 times less than that of the 99-bp
fragment. It should be noted, however, that the relative abundance of
both the actin 99 bp and 291 bp did not change significantly over the
12-hour time series.
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To test the effect of pre-fixation time on relative representation of
mRNA species as measured by quantitative RT-PCR, we obtained a piece of
human liver tissue directly from the operating room and processed it as
described in Materials and Methods. Several genes were studied and the
results for GAPDH, ZNF217, ErbB-2, VEGF, Flt, and KDR are
shown in Figure 4
. All expression is relative to ß-Gus and the data are normalized to
the results for fresh tissue RNA to allow easy visualization of the
effect of the 18-hour fixation and of pre-fixation time on mRNA
quantitation. For c-myc, three different fragments of the
mRNA were assayed (Figure 5)
to study the effect of fixation and pre-fixation time on recovery and
quantitation of this highly unstable mRNA species.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously published protocols for isolating RNA from fixed
tissues20
were modified to optimize RNA yield and size.
Reverse transcription procedures were also optimized to obtain
reproducible reverse transcription efficiencies and therefore accurate
quantitation of mRNA expression levels. Despite these optimized
procedures, it was found that the absolute CT
values on fixed tissues were an average of 5 cycles higher than on
matched fresh tissues. This indicates that only one-thirtieth of the
RNA in the reaction is accessible to cDNA synthesis. Presumably the
remaining RNA is chemically altered by the formalin-fixation and
paraffin-embedding and cannot be reverse transcribed. Furthermore, the
process of formalin fixation and paraffin embedding seems to
differentially affect the ability to recover different mRNA species or
fragments of those species. This difference can be seen by looking at
expression measured in the fresh tissue RNA and the zero-hour fixed
tissue RNA, and is especially pronounced (10-fold change) in the case
of the c-myc exon 2/3 fragment relative to ß-Gus (Figure 5)
. In this case formalin fixation and paraffin embedding appears to
reduce the amount of this particular 86-bp fragment of c-myc
mRNA that can be recovered, reverse transcribed, and quantitated,
relative to the 81-bp fragment of ß-Gus. This 10-fold change is
presumably not due to biological degradation of c-myc
message, since both tissues were processed immediately on excision from
the patient and the tissue pieces were small enough (<1
cm3) for fixation to be very fast. Furthermore,
when different fragments of c-myc mRNA were analyzed
(c-myc exon 2 or c-myc 3'UTR) the relative
recovery of each fragment was closer to that from fresh tissue (Figure 5)
. Thus the ability to recover and reverse transcribe different
fragments of one mRNA species varies from fragment to fragment,
possibly as a result of regulatory proteins bound to (and then
cross-linked to) different parts of the mRNA.34
Despite the improved RNA isolation protocol, RNA obtained from fixed
tissues was still highly degraded, and thus one would expect smaller
amplicon sizes to provide better sensitivity. When this was tested, we
found that there was an increase in the CT value
as amplicon size increased and that this increase was much greater with
fixed tissue RNA than with control, fresh tissue RNA. The delta
CT for the ß-actin 291-bp versus the
99-bp (CT291–99) product was 3.4 cycles on
the fresh tissue RNA and 9.9 cycles on the immediate fix RNA sample. By
calculating a CT for the fixed
tissue RNA minus the fresh tissue RNA
(CT291–99 fresh tissue -
CT291–99 fixed tissue = 6.5) to correct
for PCR efficiency differences, it can be determined that the relative
abundance of the 99-bp and 291-bp fragments in the fixed tissues is
90:1 (26.5:1). Furthermore, the absolute
CT values on fixed tissue RNA did not change
significantly with fragment sizes <131 bp, but then increased by 5
cycles for the 175-bp fragment. Thus, it seems that the best
sensitivity for RT-PCR on fixed tissue RNA can be obtained with
amplicon sizes less than 130 bp. No large difference was seen
between the zero-hour and 12-hour pre-fixation time data points,
indicating that RNA degradation and cross-linking due to the fixation
and embedding procedure is worse than any degradation occurring
in the tissue before fixation. Therefore, comparing fragments of
different sizes should give a constant ratio, as was the case when we
studied 99-bp and 291-bp fragments of the ß-actin gene (Figure 3)
,
but this has not been thoroughly tested, so it seems prudent to keep
both test and reference amplicons below 130 bp.
For all quantitative analyses, three separate cDNA synthesis reactions
were carried out with different RNA input amounts (75–300 ng). We
believe that this has several advantages over single RNA input
quantitation. First, by analyzing three points, one should see a linear
decrease in CT that corresponds to increasing RNA
input in the RT reaction. If linearity is not observed, then the RT
step was not quantitative, and therefore the results are questionable.
Second, the slopes of RNA input versus
CT should be close to identical for the test and
reference genes on any single RNA (and preferably on all RNAs). This
similarity in slopes indicates that both the RT and PCR efficiencies
were the same for both genes, a critical factor when using
CT calculations for
quantitation.24
The main factor influencing RT slopes
appeared to be the RNA purity, and therefore we found it necessary to
use the rather laborious RNA isolation procedures described. Third, the
use of three RT points allows one to obtain an estimate of the error
associated with the measurement. The mean and SD of the
CT values can be calculated and plotted as
in Figure 4
.
It might be expected that pre-fixation time would be the major factor influencing the ability to quantitate gene expression in fixed tissues. mRNAs with short half-lives would presumably disappear quickly, and thus pre-fixation time would affect the levels detected. Because pre-fixation time is unknown for archival tissue specimens, quantitation of expression would be impossible. Interestingly, this was not the case. Our experiments showed that times up to 12 hours in PBS before fixation and embedding did not change the relative expression of any of the genes studied using this technique. Even c-myc mRNA, which has a reported half-life as short as 10 minutes,35 did not show a reduction with up to 12 hours pre-fixation time. This is probably due to two factors. First, the RT-PCR assay only requires a small fragment of intact RNA, so the RNA can be substantially degraded and still be detected. Second, it is reasonable to assume that the tissue is still metabolizing. mRNA half-lives are typically determined by using actinomycin D to stop RNA synthesis and then measuring decay of the pre-existing mRNA over time. In vivo, however, steady state levels of mRNA are controlled by both transcription and degradation rates.36, 37 Assuming that transcription is still occurring in the tissues incubated in PBS, there is no a priori reason to expect steady-state levels to change. In support of this assumption, when we carried out a pre-fixation time study using a piece of tissue that had been stored at -70°C instead of collected directly from the operating room, the c-myc mRNA levels dropped dramatically over an 8-hour pre-fixation time course (data not shown). This implies that, once frozen, tissue metabolism is stopped and steady-state levels of mRNA are then influenced only by degradation rates, thus the c-myc mRNA disappears quickly. Of course, for many genes transcription and degradation rates will be affected by stresses such as, eg, hypoxia,38, 39, 40 temperature reduction,41 and depletion of nutrients42, 43 imposed by excision and storage at room temperature in PBS. Indeed, in our experiments, GAPDH levels increased relative to ß-Gus over the 12-hour study period, possibly due to hypoxic induction of GAPDH.44, 45 For some genes these expression changes will be considerable and may preclude meaningful quantitation from fixed tissues collected in an uncontrolled manner. By carrying out a pre-fixation time study such as the one described here, one should be able to determine whether a particular mRNA of interest is subject to change with pre-fixation time and thus validate that gene for fixed tissue expression studies. We have studied >10 genes so far and only GAPDH shows a significant trend with time; even this results in a change of less than a factor of two over 12 hours. Because it is unlikely that tissue would be left more than 12 hours from surgical excision to formalin fixation, it should be possible to go back to archival fixed tissue banks and obtain meaningful gene expression data using TaqMan quantitative RT-PCR.
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Footnotes |
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Supported by Specialized Program of Research Excellence grant CA 58207 and by National Institutes of Health grant CA 84019. S.-H. K. was supported by the Korean Research Foundation.
Accepted for publication February 25, 2000.
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T. M Schaefer, J. A. Wright, P. A. Pioli, and C. R. Wira IL-1{beta}-Mediated Proinflammatory Responses Are Inhibited by Estradiol via Down-Regulation of IL-1 Receptor Type I in Uterine Epithelial Cells J. Immunol., November 15, 2005; 175(10): 6509 - 6516. |
