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Technical Advance |
From the Institute of Pathology,*
GSF-National
Research Center for Environment and Health, Neuherberg; and the
Institute of Pathology,
Technical University
Munich, Munich, Germany
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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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Real-time quantitative TaqMan reverse transcriptase-polymerase chain reaction (QRT-PCR) analysis has recently been introduced as a sensitive, accurate, and highly reproducible method to study gene expression. The technique is based on the 5' nuclease activity of Taq DNA polymerase and involves cleavage of a specific fluorogenic hybridization probe that is flanked by PCR primers spanning an amplicon range of 60 to 150 bp.9,10 Because of the small target size, this approach seemed to be particularly suitable for quantitative determination of gene transcript levels even in tissue extracts containing partially fragmented RNA. As this experimental strategy requires only minute amounts of RNA it should be applicable to small clinical biopsies and microdissected cell clusters from frozen or formalin-fixed, paraffin-embedded tissue sections. Laser-assisted microdissection has become indispensable for the selective analysis of stroma-free tumor cell populations circumventing the problem of tissue heterogeneity as well as providing the possibility to assign characteristic gene expression patterns to particular histological phenotypes.11,12
Here we present a new approach toward the detection and quantitation of specific mRNA levels in formalin-fixed, paraffin-embedded samples that combines real-time QRT-PCR and laser-assisted microdissection. Using a panel of cancer-relevant genes, we performed quantitative gene expression analysis in matched frozen and formalin-fixed, paraffin-embedded tissue samples. We examined the influence of different parameters on reliable and reproducible mRNA quantitation, including several microscale RNA extraction protocols, formalin-fixation time and laser microdissection of defined numbers of cells. Finally, we analyzed 54 microdissected nonneoplastic and neoplastic samples from 26 archival adenocarcinoma of the esophagus by QRT-PCR for HER-2/neu gene expression and compared the results with those obtained by fluorescence in situ hybridization (FISH) and immunohistochemistry.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human A431 epidermoid carcinoma cells (CRL-1555; American Type Culture Collection, Rockville, MD) were grown in Dulbecco’s minimal essential medium supplemented with 2 mmol/L L-glutamine and 10% fetal calf serum. Human HT29 colon adenocarcinoma cells (HTB38; American Type Culture Collection) were grown in McCoy’s medium supplemented with 2 mmol/L glutamine and 10% fetal calf serum. Media and supplements were purchased from Gibco BRL (Eggenstein, Germany).
Tumor Formation in Nude Mice
Five-week-old athymic (nu/nu) mice were obtained from Charles River Breeding Laboratories (Wilmington, MA). Cultured A431 and HT29 tumor cells were resuspended in 100 µl of sterile phosphate-buffered saline at a cell density of 2 x 106 and injected subcutaneously into the flank region of nude mice. Tumor formation was monitored twice weekly by measuring the width and length of the tumors. Animals with mean tumor diameters of 15 mm were sacrificed and tumor samples were cut in halves. Half of each of the tumors was fixed in 10% buffered formalin for 24 hours within 1 hour after surgical removal and paraffin-embedded using standard procedures, the other half of the tumor was immediately snap-frozen and stored in liquid nitrogen until use.
Tissue Samples
For testing the specificity of the QRT-PCR and variables of the formalin fixation procedure, liver, uterus with leiomyoma, and a prostate cancer specimen obtained from three different patients were used. The tissue samples were fixed in 10% neutral-buffered formalin for 20 hours within 1 hour after surgical removal and paraffin-embedded using standard procedures. For the HER-2/neu analyses, archival material from formalin-fixed, paraffin-embedded tissue obtained from 26 patients with primary Barrett’s adenocarcinoma of the distal esophagus was used. Serial sections were cut for immunohistochemistry (5 µm), FISH (10 µm), and quantitative real-time RT-PCR analysis (5 µm). Corresponding areas on sequential sections were investigated by the three methods.
Tissue Preparation and Microdissection
Using RNase-free conditions, frozen tissue blocks were sectioned
at 5 µm in a cryostat, mounted on noncoated clean glass slides, and
stored at -80°C until use. Formalin-fixed, paraffin-embedded tissue
samples were cut in 5-µm-thick sections on a microtome with a
disposable blade. For microdissection, sections were deparaffinized in
two changes of xylene for 10 minutes, rehydrated in 100% ethanol, 90%
ethanol, and 70% ethanol for 5 minutes each, stained with hematoxylin
and eosin (H&E) for 45 seconds, rinsed in RNase-free
H2O for 30 seconds, and finally immersed in 100%
ethanol for 1 minute. The PALM Laser-MicroBeam System (P.A.L.M.,
Wolfratshausen, Germany) was used for microdissection. This system
consists of a 337-nm pulsed nitrogen laser coupled to an inverted
microscope via the epifluorescence illumination path. After selecting
the cells of interest, adjacent cells were photolysed by the microbeam.
To retrieve the selected cells from the slide, a computer-controlled
micromanipulator and conventional sterile needles were used to pick and
transfer the cells into a reaction tube as described
previously.11
For quantitative gene expression analyses in
formalin-fixed, paraffin-embedded A431 and HT29 xenografts, groups of
tumor cells (ncell = 
10,000, 1,000, 100,
and 50) were laser-microdissected from H&E-stained 5-µm sections. For
the HER-2/neu gene expression analyses in Barrett’s
adenocarcinoma, groups of 1,000 cells were microdissected from
nonneoplastic squamous epithelium or gastric mucosa and carcinoma
lesions, respectively.
Extraction of Total RNA from Formalin-Fixed, Paraffin-Embedded Tissue
Total RNA from liver, uterus, leiomyoma, the prostate cancer specimen, and A431 and HT29 tumor xenografts was extracted from formalin-fixed, paraffin-embedded tissue using a modification of the method described by Rupp and Locker.6 Briefly, for the analysis of unstained 5-µm sections, tissue was scraped off and paraffin was removed by extracting two times with 1 ml of xylene for 10 minutes followed by rehydration through subsequent washes with 100, 90, and 70% ethanol diluted in RNase-free water. After each step, the tissue was collected by centrifugation at 16,000 x g for 5 minutes. After the final 70% ethanol wash, the pellet was dried, resuspended in 200 µl of RNA lysis buffer containing 10 mmol/L Tris/HCL (pH 8.0), 0.1 mmol/L ethylenediaminetetraacetic acid (pH 8.0), 2% sodium dodecyl sulfate (pH 7.3), and 500 µg/ml proteinase K (Sigma, Deisenhafen, Germany) and incubated at 60°C for 16 hours until the tissue was completely solubilized. Alternatively, for the microdissection experiments with A431 and HT29 tumor xenografts and for the HER-2/neu gene expression analyses in Barrett’s adenocarcinoma, microdissected cells from H&E-stained sections were directly transferred into a sterile 1.5-ml tube and lysed in 200 µl of RNA lysis buffer. RNA was purified by phenol and chloroform extractions followed by precipitation with an equal volume of isopropanol in the presence of 0.1 volume of 3 mol/L sodium acetate (pH 4.0), and 1 µl of 10 mg/ml of carrier glycogen at -20°C. The RNA pellet was washed once in 70% ethanol, dried, and resuspended in 10 µl of RNase-free water. In comparing experiments, RNA was isolated from formalin-fixed, paraffin-embedded liver tissue using either the method described above with Proteinase K digestion at 60°C and 42°C or three different protocols13-15 after tissue sections had been deparaffinized as described above.
Extraction of RNA from Frozen Tissue
Total RNA was extracted from scraped frozen tissue sections with the Micro RNA Isolation Kit (Stratagene, San Diego, CA) following the manufacturer’s protocol.
Reverse Transcription
RNA extracted from frozen and formalin-fixed, paraffin-embedded tissue sections was reverse-transcribed in a final volume of 20 µl using M-MLV reverse transcriptase (Gibco-BRL) in the manufacturer’s buffer containing 1 mmol/L dNTPs, 40 U of RNase inhibitor (Amersham Pharmacia Biotech, Freiburg, Germany), 300 ng of random hexamers (Pharmacia), and 7 µl of RNA. The reactions took place at 42°C for 60 minutes, followed by 95°C for 5 minutes and 4°C for 5 minutes.
Real-Time Quantitative RT-PCR
Real-time quantitative RT-PCR analyses for EGF-R,
HER-2/neu, FGF-R4, p21/WAF1/Cip1,
MDM1, HPRT, and PGK mRNAs were
performed using the ABI PRISM 7700 Sequence Detection System instrument
and software (PE Applied Biosystems, Inc., Foster City, CA).
Intron-spanning primers and probes for the TaqMan system were designed
to meet specific criteria by using Primer Express software (Perkin
Elmer, Foster City, CA) and were synthesized by PE ABI (Weiterstadt,
Germany). The 5'- and 3'-end nucleotides of the probe were labeled with
a reporter (FAM = 6-carboxy-fluorescein) and a quencher dye
(TAMRA = 6-carboxy-tetramethylrhodamine). The sequences of the PCR
primer pairs and fluorogenic probes that were used for each gene are
shown in Table 1
. The oligonucleotides
are designated by the nucleotide position relative to EGF-R
GenBank accession no. X00588, HER-2/neu GenBank accession
no. M11730, FGF-R4 GenBank accession no. L03840,
p21/WAF1/Cip1 GenBank accession no. L25610, MDM2
GenBank accession no. M92424, HPRT GenBank accession no.
M31642, TBP (a component of the DNA-binding protein complex
TFIID) GenBank accession no. X54993 and PSA
(prostate-specific antigen) GenBank no. X05332. PGK primers
and probes were purchased from Perkin-Elmer. The principle of real-time
RT-PCR has been described in detail elsewhere.9,10
Briefly, real-time RT-PCR is based on fluorescence emission by a
sequence-specific, nonextendable probe labeled with a 5'-reporter and
3'-quencher dye. During the extension phase of the PCR, the nucleolytic
activity of the Taq DNA polymerase cleaves the hybridization
probe and the subsequent separation of quencher and reporter dye
releases a fluorescence signal that is monitored every 8.5 seconds by a
sequence detector. The signal is normalized to an internal reference
(
Rn) and the software sets the threshold cycle Ct, when Rn
becomes equal to 10 standard deviations of the baseline. Ct is used for
quantitation of the input target number. The relative expression level
of the gene of interest was computed with respect to the internal
standard, PGK to normalize for variances in the quality of RNA and the
amount of input cDNA. For each experimental sample, the amount of
target and endogenous reference was determined from a standard curve.
The latter was constructed with fivefold serial dilutions of A431
carcinoma cell line cDNA (100,000 pg to 16 pg) and was run in duplicate
during every experiment. The amount of target gene was divided by the
endogenous reference amount to obtain a normalized target value. PCR
was performed with the TaqMan Universal PCR Master Mix (PE, Applied
Biosystems) using 3 to 5 µl of diluted cDNA, 200 nmol/L of the probe,
and 300 nmol/L primers (except HER-2/neu RP and FGF-R4 FP,
which were used at 50 nmol/L and 900 nmol/L, respectively) in a 30-µl
final reaction mixture. After a 2-minute incubation at 50°C to allow
for UNG cleavage, AmpliTaq Gold was activated by an incubation for 10
minutes at 95°C. Each of the 50 PCR cycles consisted of 15 seconds of
denaturation at 95°C and hybridization of probe and primers for 1
minute at 60°C.
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For FISH analysis a PathVysion HER-2/neu DNA probe kit (Vysis, Inc., Downer’s Grove, IL) was used according to the manufacturer’s recommendation. Signals from 100 to 150 tumor cell nuclei were counted using confocal laser-scanning microscopy (Zeiss LSM 410). According to published criteria,16 gene amplification was detected if the ratio of locus-specific signals to centromeric signals per cell was at least three in >10% of tumor cells, or tight clusters of >10 locus-specific signals occurred in multiple cells.
Immunohistochemistry
HER-2/neu protein expression was assessed on paraffin-embedded tissue samples using the anti-c-erbB-2 antibody (DAKO, Glostrup, Denmark) with a DAKO ChemMate detection kit according to the manufacturer’s recommendation. To ensure the sensitivity of the reaction in all cases, immunoreactive tissue of an invasive ductal breast carcinoma with known overexpression of c-erbB-2 was used as positive control. HER-2/neu protein overexpression was evaluated using a light microscope according to a score system as recommended by the DAKO HercepTest. Briefly, no staining at all or membrane staining in <10% of the tumor cells were scored as 0; a barely perceptible membrane staining in >10% of the tumor cells was scored as 1+; a weak-to-moderate staining of the entire membrane in >10% of the tumor cells was given a score of 2+; and a strong staining of the entire membrane in >10% of the tumor cells was scored as 3+.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As a first step toward the establishment of a quantitative gene
expression methodology applicable to formalin-fixed, paraffin-embedded
tissue, we examined RNA extracted by five different procedures from
single routine paraffin sections by real-time TaqMan RT-PCR analysis.
The expression of five cancer-relevant genes, EGF-R,
HER-2/neu, FGF-R4, p21/WAF1/Cip1, and
MDM2 with mRNA half-lives ranging from 2 to 10 hours
(unpublished results) was investigated.17
All primers and
hybridization probes were designed to span an intron to exclude
annealing to genomic DNA and amplicon sizes were kept small (66 to 93
bp; see Table 1
). PGK as well as HPRT were
included as housekeeping gene controls to correct for variations in the
degree of RNA degradation and efficiencies of RNA extraction and
reverse transcription. After optimization of the experimental
conditions for the seven primer/probe combinations, controls were
performed to demonstrate the specificity of the RT-PCR reaction. No
signals were obtained with genomic DNA or when reverse transcriptase or
RNA were omitted (data not shown). Reproducibility and sensitivity of
the extraction procedure and subsequent quantitative real-time RT-PCR
amplification of the seven target sequences was assessed by different
parameters of the TaqMan assay, such as low Ct and high Rn values
(data not shown). Proteinase K digestion at high temperature followed
by organic extraction (described in Materials and Methods) yielded the
highest RNA amounts and the most reproducible results as compared to
RNA extraction using Proteinase K digestion at 42°C, acidic
guanidinium thiocyanate-phenol chloroform,13
oligo d(T)
coupled magnetic beads,14
or guanidinium thiocyanate lysis
combined with a silica-matrix spin technology15
as
shown representatively for the p21/WAF1/Cip1 Ct values (Table 2)
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To investigate gene expression in matched frozen and
formalin-fixed, paraffin-embedded tissue, we used nude mice
subcutaneous tumor xenografts of human colon adenocarcinoma HT29
(Figure 1A)
and epidermoid carcinoma A431
cell lines because of their homogeneous histological characteristics.
Isolation of RNA from 5-µm sections of A431 and HT29 tumors yielded
only slightly more material for frozen than for formalin-fixed,
paraffin-embedded tissue material. On serial dilutions of both
preparations, the RNA was reverse-transcribed and real-time
amplification of EGF-R, HER-2/neu,
FGF-R4, p21/WAF1/Cip1, MDM2,
PGK, and HPRT sequences was performed. In all
cases, there was a strong linear correlation between the number of
thermal cycles required to generate a significant fluorescent signal
above background and the log of the input cDNA amount
(R2
0.99). Quantitative measurements could be
made over an extremely wide range of target concentration (16 pg to 50,
000 pg). Even more importantly, when the resulting Ct values were
plotted against the log of the initial template amount and subjected to
linear regression analysis, the amplification efficiencies were found
to be very similar in formalin-fixed, paraffin-embedded and frozen
tissue xenografts as representatively shown for the EGF-R
gene in HT29 tumors (Figure 1, B and C)
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, there was
no significant difference between gene expression levels obtained with
frozen or formalin-fixed, paraffin-embedded xenograft sections.
Subsequently, the comparability of gene expression analysis results
from matched frozen and formalin-fixed, paraffin-embedded material was
confirmed with colorectal carcinoma samples and lymph node metastases
from four patients, further demonstrating the value of the procedure
for clinical research (data not shown).
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, shows that amplicon sizes up
to 122 bp gave the best results for FFPE tissue as indicated by low Ct
values, whereas no cDNA product was obtained with primers that were 374
bases apart. When template from matching frozen control tissue was used
under the same reaction conditions, PCR products up to 374 bases were
easily detectable.
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To address parameters of the formalin fixation procedure, which
depending on the clinical setting may influence gene expression
measurements, we asked whether fixation grade can affect mRNA
quantitation. Because tissue infiltration by the fixative is a slow
process, extensive degradation of RNA may occur in the center of big
specimens. We therefore performed quantitative gene expression analysis
in big (>7 cm) tissue samples with decreasing grades of fixation
toward the center. Because of their morphological homogeneity, we
analyzed liver and uterus with leiomyoma tissues that had been fixed in
formalin for 20 hours. Sequential sections of 1 cm were cut to a depth
of 3 to 6 cm, and the blocks were paraffin-embedded using standard
procedures. From every single cm of this tissue, RNA extraction was
performed to measure gene expression of the five markers and two
control genes to analyze whether differences in fixation might
influence RNA expression measurements. The results of the relative gene
expression measurements were found not to be significantly different
with the exception of p21/WAF1/Cip1, which was up-regulated nearly
11-fold in leiomyoma tissue as compared to the adjacent myometrium. The
latter observation was confirmed by immunohistochemical analysis (data
not shown) suggesting that this finding was not caused by an intrinsic
methodological artifact. Relative measurements and absolute Ct values
for the PGK and HPRT housekeeping genes are shown
in Figure 4
; A, B, and C.
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To further demonstrate the specificity of the approach,
prostate-specific antigen (PSA) mRNA amounts were measured
in a formalin-fixed, paraffin-embedded prostate cancer specimen and in
liver, myometrium, and leiomyoma tissues. As expected, the measurement
of PSA mRNA resulted only in a signal with RNA isolated from
the prostate cancer specimen and not with RNA isolated from the control
tissues (Figure 5B)
. In contrast, the
TBP mRNA that was determined as a positive control was
readily detectable in all four tissues examined as shown in the
amplification plots in Figure 5A
and the corresponding analytical
agarose gel in Figure 5C
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Laser-assisted microdissection has emerged as an important
technique for the analysis of morphologically defined areas of tissue
sections. To examine the applicability of our approach to such small
tissue samples, we microdissected cell clusters consisting of
10,000, 1,000, 100, and 50 tumor cells from H&E-stained
formalin-fixed, paraffin-embedded A431 and HT29 xenograft sections and
determined gene expression levels in relation to a complete 5-µm
section containing 106
cells. Three
independent experiments involving separate cell picking, RNA
extraction, and reverse transcription were performed and expression of
the seven different transcripts were examined from the same reverse
transcription reaction. As shown in Figure 6
, mRNA level determinations were
comparable within the range of 106
to 100 cells
for all six genes analyzed, demonstrating accuracy and reproducibility
of the procedure. For FGF-R4 and MDM2 genes,
expression in 50 microdissected cells from HT29 xenografts was at the
borderline of detection, whereas the remaining genes of our panel
yielded values comparable to all other preparations.
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To further apply our approach in a clinically relevant context, we
measured HER-2/neu gene expression in 54
laser-microdissected formalin-fixed, paraffin-embedded samples of 26
patients with esophageal adenocarcinoma. Real-time TaqMan RT-PCR
performed on RNA extracted from laser-microdissected tumors
(n = 26) revealed relative HER-2/neu
mRNA expression levels from 0.27 to 83.57, whereas the surrounding
normal squamous epithelium (n = 17) and gastric
mucosa (n = 11) showed expression levels in a
range of 0.26 to 1.7 (mean, 0.88 ± 0.38) and 0.36 to 2.7 (mean,
1.03 ± 0.59), respectively. Numerous studies show that
amplification of the HER-2/neu gene and overexpression of
the HER-2/neu protein closely parallel HER-2/neu mRNA
overexpression.18
We therefore compared the values
generated by real-time QRT-PCR to corresponding DNA and protein data
obtained by FISH and immunohistochemistry conducted on adjacent serial
sections of the same tissue blocks. Among the 26 adenocarcinoma samples
tested, 10 cases (38%) were found to have both an amplified
HER-2/neu gene and to overexpress the HER-2/neu protein
(except one case, no. 5, which displayed very weak HER-2/neu protein
expression despite high-level HER-2/neu gene amplification).
All of these 10 cases show significantly higher levels of
HER-2/neu mRNA in the tumor than in the surrounding normal
tissue (Table 3)
. The other 16 cases
without HER-2/neu gene amplification showed relative
HER-2/neu mRNA expression levels in the range of 0.27 to 1.1
(data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The aim of this study was to develop an experimental procedure that would allow an accurate, reproducible, quantitative, high-throughput analysis of specific mRNA levels in standard paraffin-embedded pathology specimens.
Previous studies reporting molecular analyses of RNA from formalin-fixed, paraffin-embedded tissue present findings of a mostly qualitative nature, such as the detection of viral nucleic acids in human tissues, or identification of tumor-specific products of chromosomal translocations and results of gene mutations.20-22 Because of the lack of methodologies that allow accurate and simple quantitative measurements, there are only very few studies that report attempts of quantitative gene expression analysis in archival tissues to date.23 A variety of approaches routinely used to assess the expression of specific genes in cells or tissues, such as Northern blot-, RNase protection-, S1 nuclease-, or in situ hybridization analysis have considerable drawbacks when applied to formalin-fixed, paraffin-embedded tissue sections: either the fragmented nature of the RNA precludes the application of these techniques or they may not be performed with small tissue samples, clinical biopsies, and microdissected cell clusters. Moreover, the sensitivity and accuracy of some of these techniques is limited. In contrast, the recently developed real-time quantitative RT-PCR is an easy, versatile, sensitive as well as accurate and precise method for the study of gene expression.9,10 Real-time systems are capable of detecting PCR products as they accumulate during amplification, and the reactions are characterized by the point during cycling when PCR amplification is still in the exponential phase, thus enabling precise quantitation of RNA throughout a wide dynamic range. As little as 10 copies of a specific transcript can be detected and quantified. Moreover, the assay is compatible with high-throughput analysis, allowing 96 samples to be analyzed in only 2 hours.
We used real-time QRT-PCR based on TaqMan methodology to
quantitatively analyze gene expression in routinely processed
formalin-fixed, paraffin-embedded tissues and demonstrate that gene
expression measurements can be reliably and accurately conducted in
such tissues. A prerequisite for our gene expression studies in
formalin-fixed tissue samples was the establishment of a reliable and
reproducible microscale RNA extraction method in conjunction with
reverse transcription that would provide an optimal cDNA as template
for real-time PCR amplification. To date, various methods have been
used for the isolation of RNA from archival formalin-fixed tissues and
we compared five of the most commonly used ones in conjunction with
TaqMan RT-PCR as quality control.6,13-15
Our experiments
showed that proteinase K digestion followed by organic extraction of
the RNA yields the best results in terms of both reliability and
sensitivity of the subsequent QRT-PCR (Table 2)
. It seemed that the
protease was capable of efficiently degrading proteins that were
covalently cross-linked with each other and the RNA, thereby allowing
more efficient RNA extraction than chaotropic agents. In line with
observations reported by Masuda and colleagues,8
incubation of the tissue extracts or the already extracted RNA at high
temperature (60 to 70°C) proved to be another important parameter,
possibly by reversing the methylol additions induced by the formalin
fixation and thereby improving reverse transcription efficiency. Most
importantly, amplification protocols that involved only small RNA
target sequences proved to be most successful presumably because of the
significantly reduced risk of cross-link occurrence in the region
bordered by the PCR primers (Figure 3, A and B)
confirming an
observation that previously has been made by Goldsworthy and
colleagues.24
Using the proteinase K digestion method, we did not find significant
differences in the amount of extracted RNA between fixed and
fresh-frozen 5-µm sections (Figure 1, B and C)
. Indeed, our
experiments with matched frozen and formalin-fixed, paraffin-embedded
tissue from human tumor cell-line xenograft model tissues demonstrated
that gene expression level measurements were in a comparable range
(Figure 2)
. Regardless of the quantity of extractable RNA from
formalin-fixed tissues and assuming that the degree of cross-linkage
induced by the formalin fixation is similar in all mRNAs of a given
sample, the use of a housekeeping gene as an internal reference
standard is of great importance. This allows the accurate control of
cDNA amounts as well as the quality of the extraction and reverse
transcription steps thereby ensuring reliable mRNA quantitation.
Previous studies concluded that RNA in formalin-fixed,
paraffin-embedded tissues undergoes significant enzymatic
degradation6,24-26
because of the different length of
time until the specimens are fixed in formalin after surgical removal
or because of variable influences during the processing of the tissues
until complete fixation and embedding is achieved. Interestingly, our
investigation of parameters relevant in this context indicate that
within a period of 20 hours, the extent of formalin fixation has no
major influence on the suitability of RNA from paraffin-embedded
tissues for real-time QRT-PCR analysis (Figure 4)
, whereas we cannot
rule out the possibility that the degree of RNA degradation differs
under certain fixation conditions and may vary from tissue to tissue.
However, even in a morphologically homogenous, RNase-rich tissue such
as liver, transcript levels of the seven genes with mRNA half-lives
ranging from 2 to 10 hours were not significantly altered in the
different tissue layers (Figure 4A)
. We therefore conclude that
although the RNA may undergo degradation, the resulting fragments are
still large enough to be detected by TaqMan QRT-PCR. Seemingly, our
findings are in disagreement with other reports; however, the amplicon
sizes we choose were much smaller than those used in other
studies.7,20,21
Moreover we only used resected surgical
specimens that had been fixed within 1 to 2 hours after removal and had
been subject to a formalin fixation duration of a maximum of 72 hours.
Until now, we successfully extracted and analyzed RNA from >250
formalin-fixed, paraffin-embedded tumor tissue blocks including some
>20 years old. In all cases, we were able to extract amplifiable RNA
with HPRT and PGK as control mRNA targets, suggesting that the time
embedded in paraffin did not have an effect on the RNA. It should be
noted, nonetheless, that the paraffin blocks were always sealed after
preparation of slides, thus minimizing the risk of oxidization of
tissue.
A procedure for reliable quantitative measurements of gene expression
in archival tumor tissue is one important aspect when addressing basic
questions regarding specific disease mechanisms; another critical point
refers to the accurate access of cells to be examined.27
To generate contamination-free, homogenous tumor cell populations,
laser-microdissection has been introduced and is now generally accepted
as a powerful tool to dissect morphologically identified cell
populations.11,12
Our laser microdissection studies with
defined numbers of cells as starting material for QRT-PCR showed that
quantitative gene expression measurements can be performed in as few as
50 microdissected cells from formalin-fixed, paraffin-embedded tissue
sections (Figure 6)
. To our knowledge, this is the first report
demonstrating that relative mRNA transcript levels can be reliably
determined from such small cell numbers. Schütze and
colleagues28
reported single-cell RT-PCR from archival
tissue however they used a nested RT-PCR approach to qualitatively
detect c-Ki-ras2 mRNA mutations in colon adenocarcinoma. In another
recent publication, Fink and colleagues27
applied QRT-PCR
to cell clusters containing 10 to 15 cells, but they used fresh-frozen
tissue for their analyses. Fifty cells were the lowest number of cells
used in the present study, however, when amplifying highly abundant
mRNA transcripts, we even succeeded in performing single-cell real-time
QRT-PCR from formalin-fixed, paraffin-embedded tissue (data not shown).
To further prove the clinical usefulness of our procedure, we choose
HER-2/neu as the analyte prototype for validation.
Overexpression and amplification of the HER-2/neu gene is
known to be involved in many human cancers, including breast, ovarian,
and gastrointestinal carcinoma and has been shown to correlate with
tumor grade, tumor size, and disease progression.18
Among
the gastrointestinal cancers, esophageal Barrett’s adenocarcinoma
exhibits the most rapidly increasing incidence and 20 to 60% of
these adenocarcinomas show HER-2/neu gene amplification or
overexpression of the HER-2/neu protein.29-31
The results
of our investigation indicate that among the 26 adenocarcinoma tested,
10 cases (38%) have both an amplified HER-2/neu gene and/or
overexpress HER-2/neu protein and have significantly higher relative
HER-2/neu transcript levels in tumor lesions than in the
adjacent normal tissue (Table 3)
. The relative HER-2/neu
expression ranges from 0.27 to 83.57 in tumor lesions as compared to
only 0.26 to 1.7 in normal squamous epithelium, and 0.36 to 2.7 in
gastric mucosa. Overall, HER-2/neu status assessed by FISH
analysis and immunohistochemistry closely correlated with
HER-2/neu gene overexpression measured by TaqMan QRT-PCR.
Our findings in terms of the frequency of HER-2/neu
amplification in Barrett’s adenocarcinoma were in good agreement with
published data.29-31
As it is well established that
amplification of the HER-2/neu gene and overexpression of
the HER-2/neu protein closely parallel HER-2/neu mRNA
overexpression,18
these data represent a validation of our
approach and suggest that it should be broadly and generally applicable
to quantitative gene expression analysis in archival tissue.
In conclusion, we report the first application of combined QRT-PCR and laser-assisted microdissection for the quantification of gene expression levels in archival formalin-fixed, paraffin-embedded resected tissue samples. We demonstrated that mRNA levels in formalin-fixed, paraffin-embedded tissues routinely prepared from surgery and fixed with buffered formalin can be reproducibly and precisely determined and that the values obtained are comparable to matched frozen specimens. Controlled laser microdissection studies using defined numbers of cells showed that mRNA quantitation can be reliably performed from as few as 50 cells. Crucial aspects for the success of our procedure are: 1) an RNA microscale extraction protocol that provides only minimally cross-linked RNA, thus improving greatly the efficiency and the success of reverse transcription and QRT-PCR; and 2) the selection of small target sequences in a range of 60 to 100 bp, enabling the detection of fragmented and degraded RNA. Taken together, the combination of QRT-PCR and microdissection technologies with a reproducible microscale RNA extraction procedure establishes a simple, quantitative and highly accurate high-throughput procedure that allows retrospective studies and correlation of gene expression with clinicopathological features of large series of archival paraffin-embedded tumor tissue.
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Acknowledgements |
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Footnotes |
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Accepted for publication November 10, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R. Langer, K. Ott, K. Specht, K. Becker, F. Lordick, M. Burian, K. Herrmann, A. Schrattenholz, M. A. Cahill, M. Schwaiger, et al. Protein Expression Profiling in Esophageal Adenocarcinoma Patients Indicates Association of Heat-Shock Protein 27 Expression and Chemotherapy Response Clin. Cancer Res., December 15, 2008; 14(24): 8279 - 8287. [Abstract] [Full Text] [PDF]
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M. Doleshal, A. A. Magotra, B. Choudhury, B. D. Cannon, E. Labourier, and A. E. Szafranska Evaluation and Validation of Total RNA Extraction Methods for MicroRNA Expression Analyses in Formalin-Fixed, Paraffin-Embedded Tissues J. Mol. Diagn., May 1, 2008; 10(3): 203 - 211. [Abstract] [Full Text] [PDF]
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C Blind, A Koepenik, M Pacyna-Gengelbach, G Fernahl, N Deutschmann, M Dietel, V Krenn, and I Petersen Antigenicity testing by immunohistochemistry after tissue oxidation J. Clin. Pathol., January 1, 2008; 61(1): 79 - 83. [Abstract] [Full Text] [PDF]
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T. Haque, D. Faury, S. Albrecht, E. Lopez-Aguilar, P. Hauser, M. Garami, Z. Hanzely, L. Bognar, R. F. Del Maestro, J. Atkinson, et al. Gene Expression Profiling from Formalin-Fixed Paraffin-Embedded Tumors of Pediatric Glioblastoma Clin. Cancer Res., November 1, 2007; 13(21): 6284 - 6292. [Abstract] [Full Text] [PDF]
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S. Lassmann, Y. Shen, U. Jutting, P. Wiehle, A. Walch, G. Gitsch, A. Hasenburg, and M. Werner Predictive Value of Aurora-A/STK15 Expression for Late Stage Epithelial Ovarian Cancer Patients Treated by Adjuvant Chemotherapy Clin. Cancer Res., July 15, 2007; 13(14): 4083 - 4091. [Abstract] [Full Text] [PDF]
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J. M. Bock, S. G. Menon, L. L. Sinclair, N. S. Bedford, P. C. Goswami, F. E. Domann, and D. K. Trask Celecoxib Toxicity Is Cell Cycle Phase Specific Cancer Res., April 15, 2007; 67(8): 3801 - 3808. [Abstract] [Full Text] [PDF]
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R. A. Coudry, S. I. Meireles, R. Stoyanova, H. S. Cooper, A. Carpino, X. Wang, P. F. Engstrom, and M. L. Clapper Successful Application of Microarray Technology to Microdissected Formalin-Fixed, Paraffin-Embedded Tissue J. Mol. Diagn., February 1, 2007; 9(1): 70 - 79. [Abstract] [Full Text] [PDF]
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R. Reiter, P. Gais, U. Jutting, M. K. Steuer-Vogt, A. Pickhard, K. Bink, S. Rauser, S. Lassmann, H. Hofler, M. Werner, et al. Aurora Kinase A Messenger RNA Overexpression Is Correlated with Tumor Progression and Shortened Survival in Head and Neck Squamous Cell Carcinoma Clin. Cancer Res., September 1, 2006; 12(17): 5136 - 5141. [Abstract] [Full Text] [PDF]
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S. Uccella, R. Cerutti, D. Vigetti, D. Furlan, R. Oldrini, I. Carnevali, G. Pelosi, S. La Rosa, A. Passi, and C. Capella Histidine Decarboxylase, DOPA Decarboxylase, and Vesicular Monoamine Transporter 2 Expression in Neuroendocrine Tumors: Immunohistochemical Study and Gene Expression Analysis J. Histochem. Cytochem., August 1, 2006; 54(8): 863 - 875. [Abstract] [Full Text] [PDF]
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D. Talantov, J. Baden, T. Jatkoe, K. Hahn, J. Yu, Y. Rajpurohit, Y. Jiang, C. Choi, J. S. Ross, D. Atkins, et al. A Quantitative Reverse Transcriptase-Polymerase Chain Reaction Assay to Identify Metastatic Carcinoma Tissue of Origin J. Mol. Diagn., July 1, 2006; 8(3): 320 - 329. [Abstract] [Full Text] [PDF]
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K. Hamatani, H. Eguchi, K. Takahashi, K. Koyama, M. Mukai, R. Ito, M. Taga, W. Yasui, and K. Nakachi Improved RT-PCR Amplification for Molecular Analyses with Long-term Preserved Formalin-fixed, Paraffin-embedded Tissue Specimens J. Histochem. Cytochem., July 1, 2006; 54(7): 773 - 780. [Abstract] [Full Text] [PDF]
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C. Delfour, P. Roger, C. Bret, M.-L. Berthe, P. Rochaix, N. Kalfa, P. Raynaud, F. Bibeau, T. Maudelonde, and N. Boulle RCL2, a New Fixative, Preserves Morphology and Nucleic Acid Integrity in Paraffin-Embedded Breast Carcinoma and Microdissected Breast Tumor Cells J. Mol. Diagn., May 1, 2006; 8(2): 157 - 169. [Abstract] [Full Text] [PDF]
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M. A. Cobleigh, B. Tabesh, P. Bitterman, J. Baker, M. Cronin, M.-L. Liu, R. Borchik, J.-M. Mosquera, M. G. Walker, and S. Shak Tumor Gene Expression and Prognosis in Breast Cancer Patients with 10 or More Positive Lymph Nodes Clin. Cancer Res., December 15, 2005; 11(24): 8623 - 8631. [Abstract] [Full Text] [PDF]
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R. Langer, K. Specht, K. Becker, P. Ewald, M. Bekesch, M. Sarbia, R. Busch, M. Feith, H. J. Stein, J.-R. Siewert, et al. Association of Pretherapeutic Expression of Chemotherapy-Related Genes with Response to Neoadjuvant Chemotherapy in Barrett Carcinoma Clin. Cancer Res., October 15, 2005; 11(20): 7462 - 7469. [Abstract] [Full Text] [PDF]
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L. Gianni, M. Zambetti, K. Clark, J. Baker, M. Cronin, J. Wu, G. Mariani, J. Rodriguez, M. Carcangiu, D. Watson, et al. Gene Expression Profiles in Paraffin-Embedded Core Biopsy Tissue Predict Response to Chemotherapy in Women With Locally Advanced Breast Cancer J. Clin. Oncol., October 10, 2005; 23(29): 7265 - 7277. [Abstract] [Full Text] [PDF]
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S. B. Hunter, V. Varma, B. Shehata, J.D.L. Nolen, C. Cohen, J. J. Olson, and C.-Y. Ou Apolipoprotein D Expression in Primary Brain Tumors: Analysis by Quantitative RT-PCR in Formalin-fixed, Paraffin-embedded Tissue J. Histochem. Cytochem., August 1, 2005; 53(8): 963 - 969. [Abstract] [Full Text] [PDF]
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B. H.A. von Rahden, H. J. Stein, F. Puhringer, I. Koch, R. Langer, G. Piontek, J. R. Siewert, H. Hofler, and M. Sarbia Coexpression of Cyclooxygenases (COX-1, COX-2) and Vascular Endothelial Growth Factors (VEGF-A, VEGF-C) in Esophageal Adenocarcinoma Cancer Res., June 15, 2005; 65(12): 5038 - 5044. [Abstract] [Full Text] [PDF]
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R. W. Tothill, A. Kowalczyk, D. Rischin, A. Bousioutas, I. Haviv, R. K. van Laar, P. M. Waring, J. Zalcberg, R. Ward, A. V. Biankin, et al. An Expression-Based Site of Origin Diagnostic Method Designed for Clinical Application to Cancer of Unknown Origin Cancer Res., May 15, 2005; 65(10): 4031 - 4040. [Abstract] [Full Text] [PDF]
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K. Mikhitarian, W. E. Gillanders, J. S. Almeida, R. Hebert Martin, J. C. Varela, J. S. Metcalf, D. J. Cole, and M. Mitas An Innovative Microarray Strategy Identities Informative Molecular Markers for the Detection of Micrometastatic Breast Cancer Clin. Cancer Res., May 15, 2005; 11(10): 3697 - 3704. [Abstract] [Full Text] [PDF]
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A. N. Schuetz, Q. Yin-Goen, M. B. Amin, C. S. Moreno, C. Cohen, C. D. Hornsby, W. L. Yang, J. A. Petros, M. M. Issa, J. G. Pattaras, et al. Molecular Classification of Renal Tumors by Gene Expression Profiling J. Mol. Diagn., May 1, 2005; 7(2): 206 - 218. [Abstract] [Full Text] [PDF]
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R. Napieralski, K. Ott, M. Kremer, K. Specht, H. Vogelsang, K. Becker, M. Muller, F. Lordick, U. Fink, J. Rudiger Siewert, et al. Combined GADD45A and Thymidine Phosphorylase Expression Levels Predict Response and Survival of Neoadjuvant-Treated Gastric Cancer Patients Clin. Cancer Res., April 15, 2005; 11(8): 3025 - 3031. [Abstract] [Full Text] [PDF]
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A. Gloghini, B. Canal, U. Klein, L. Dal Maso, T. Perin, R. Dalla-Favera, and A. Carbone RT-PCR Analysis of RNA Extracted from Bouin-Fixed and Paraffin-Embedded Lymphoid Tissues J. Mol. Diagn., November 1, 2004; 6(4): 290 - 296. [Abstract] [Full Text] [PDF]
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 M. Taron, R. Rosell, E. Felip, P. Mendez, J. Souglakos, M. S. Ronco, C. Queralt, J. Majo, J. M. Sanchez, J. J. Sanchez, et al. BRCA1 mRNA expression levels as an indicator of chemoresistance in lung cancer Hum. Mol. Genet., October 1, 2004; 13(20): 2443 - 2449. [Abstract] [Full Text] [PDF]
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S. Halin, P. Wikstrom, S. H. Rudolfsson, P. Stattin, J. A. Doll, S. E. Crawford, and A. Bergh Decreased Pigment Epithelium-Derived Factor Is Associated with Metastatic Phenotype in Human and Rat Prostate Tumors Cancer Res., August 15, 2004; 64(16): 5664 - 5671. [Abstract] [Full Text] [PDF]
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H. Takagi, M. Shibutani, N. Kato, H. Fujita, K.-Y. Lee, S. Takigami, K. Mitsumori, and M. Hirose Microdissected Region-specific Gene Expression Analysis with Methacarn-fixed, Paraffin-embedded Tissues by Real-time RT-PCR J. Histochem. Cytochem., July 1, 2004; 52(7): 903 - 913. [Abstract] [Full Text] [PDF]
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H. Schmid, C. D. Cohen, A. Henger, D. Schlondorff, and M. Kretzler Gene expression analysis in renal biopsies Nephrol. Dial. Transplant., June 1, 2004; 19(6): 1347 - 1351. [Full Text] [PDF]
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L. M. Gjerdrum, B. S. Sorensen, E. Kjeldsen, F. B. Sorensen, E. Nexo, and S. Hamilton-Dutoit Real-Time Quantitative PCR of Microdissected Paraffin-Embedded Breast Carcinoma: An Alternative Method for HER-2/neu Analysis J. Mol. Diagn., February 1, 2004; 6(1): 42 - 51. [Abstract] [Full Text]
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M. Cronin, M. Pho, D. Dutta, J. C. Stephans, S. Shak, M. C. Kiefer, J. M. Esteban, and J. B. Baker Measurement of Gene Expression in Archival Paraffin-Embedded Tissues: Development and Performance of a 92-Gene Reverse Transcriptase-Polymerase Chain Reaction Assay Am. J. Pathol., January 1, 2004; 164(1): 35 - 42. [Abstract] [Full Text] [PDF]
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T. Goldmann, E. Vollmer, and J. Gerdes What's Cooking? Detection of Important Biomarkers in HOPE-Fixed, Paraffin-Embedded Tissues Eliminates the Need for Antigen Retrieval Am. J. Pathol., December 1, 2003; 163(6): 2638 - 2640. [Full Text]
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 D. I. Kutler, V. B. Wreesmann, A. Goberdhan, L. Ben-Porat, J. Satagopan, I. Ngai, A. G. Huvos, P. Giampietro, O. Levran, K. Pujara, et al. Human Papillomavirus DNA and p53 Polymorphisms in Squamous Cell Carcinomas From Fanconi Anemia Patients J Natl Cancer Inst, November 19, 2003; 95(22): 1718 - 1721. [Abstract] [Full Text] [PDF]
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K. Steger, L. Fink, K. Failing, R. M. Bohle, S. Kliesch, W. Weidner, and M. Bergmann Decreased protamine-1 transcript levels in testes from infertile men Mol. Hum. Reprod., June 1, 2003; 9(6): 331 - 336. [Abstract] [Full Text] [PDF]
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J-Y Scoazec Tissue and cell imaging in situ: potential for applications in pathology and endoscopy Gut, June 1, 2003; 52(90004): iv1 - 6. [Abstract] [Full Text] [PDF]
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R. Gaffney, A. Chakerian, J. X. O'Connell, J. Mathers, K. Garner, N. Joste, and D. S. Viswanatha Novel Fluorescent Ligase Detection Reaction and Flow Cytometric Analysis of SYT-SSX Fusions in Synovial Sarcoma J. Mol. Diagn., May 1, 2003; 5(2): 127 - 135. [Abstract] [Full Text] [PDF]
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L. Quintanilla-Martinez, M. Kremer, K. Specht, J. Calzada-Wack, M. Nathrath, R. Schaich, H. Hofler, and F. Fend Analysis of Signal Transducer and Activator of Transcription 3 (Stat 3) Pathway in Multiple Myeloma: Stat 3 Activation and Cyclin D1 Dysregulation Are Mutually Exclusive Events Am. J. Pathol., May 1, 2003; 162(5): 1449 - 1461. [Abstract] [Full Text] [PDF]
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J. W. U. Fries, T. Roth, H.-P. Dienes, M. Weber, and M. Odenthal A novel evaluation method for paraffinized human renal biopsies using quantitative analysis of microdissected glomeruli and VCAM-1 as marker of inflammatory mesangial cell activation Nephrol. Dial. Transplant., April 1, 2003; 18(4): 710 - 716. [Abstract] [Full Text] [PDF]
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M. Konigshoff, J. Wilhelm, R. M. Bohle, A. Pingoud, and M. Hahn HER-2/neu Gene Copy Number Quantified by Real-Time PCR: Comparison of Gene Amplification, Heterozygosity, and Immunohistochemical Status in Breast Cancer Tissue Clin. Chem., February 1, 2003; 49(2): 219 - 229. [Abstract] [Full Text] [PDF]
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M. Sanchez-Carbayo Use of High-Throughput DNA Microarrays to Identify Biomarkers for Bladder Cancer Clin. Chem., January 1, 2003; 49(1): 23 - 31. [Abstract] [Full Text] [PDF]
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E. Rosivatz, I. Becker, K. Specht, E. Fricke, B. Luber, R. Busch, H. Hofler, and K.-F. Becker Differential Expression of the Epithelial-Mesenchymal Transition Regulators Snail, SIP1, and Twist in Gastric Cancer Am. J. Pathol., November 1, 2002; 161(5): 1881 - 1891. [Abstract] [Full Text] [PDF]
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K. Specht, M. Kremer, U. Muller, S. Dirnhofer, M. Rosemann, H. Hofler, L. Quintanilla-Martinez, and F. Fend Identification of Cyclin D1 mRNA Overexpression in B-Cell Neoplasias by Real-Time Reverse Transcription-PCR of Microdissected Paraffin Sections Clin. Cancer Res., September 1, 2002; 8(9): 2902 - 2911. [Abstract] [Full Text] [PDF]
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M. Kretzler, C. D. Cohen, P. Doran, A. Henger, S. Madden, E. F. Grone, P. J. Nelson, D. Schlondorff, and H.-J. Grone Repuncturing the Renal Biopsy: Strategies for Molecular Diagnosis in Nephrology J. Am. Soc. Nephrol., July 1, 2002; 13(7): 1961 - 1972. [Abstract] [Full Text] [PDF]
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T. Ernst, M. Hergenhahn, M. Kenzelmann, C. D. Cohen, M. Bonrouhi, A. Weninger, R. Klaren, E. F. Grone, M. Wiesel, C. Gudemann, et al. Decrease and Gain of Gene Expression Are Equally Discriminatory Markers for Prostate Carcinoma : A Gene Expression Analysis on Total and Microdissected Prostate Tissue Am. J. Pathol., June 1, 2002; 160(6): 2169 - 2180. [Abstract] [Full Text] [PDF]
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S. L. Karsten, V. M. D. Van Deerlin, C. Sabatti, L. H. Gill, and D. H. Geschwind An evaluation of tyramide signal amplification and archived fixed and frozen tissue in microarray gene expression analysis Nucleic Acids Res., January 15, 2002; 30(2): e4 - e4. [Abstract] [Full Text] [PDF]
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D. D'Orazio, M. Stumm, and C. Sieber Accurate Gene Expression Measurement in Formalin-Fixed and Paraffin-Embedded Tumor Tissue Am. J. Pathol., January 1, 2002; 160(1): 383 - 384. [Full Text] [PDF]
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U. Lehmann, O. Bock, F. Langer, and H. Kreipe Demonstration of Light Chain Restricted Clonal B-Lymphoid Infiltrates in Archival Bone Marrow Trephines by Quantitative Real-Time Polymerase Chain Reaction Am. J. Pathol., December 1, 2001; 159(6): 2023 - 2029. [Abstract] [Full Text] [PDF]
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M. Schoenberg Fejzo and D. J. Slamon Frozen Tumor Tissue Microarray Technology for Analysis of Tumor RNA, DNA, and Proteins Am. J. Pathol., November 1, 2001; 159(5): 1645 - 1650. [Abstract] [Full Text] [PDF]
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 C. Maihofner, U. Schlotzer-Schrehardt, H. Guhring, H. U. Zeilhofer, G. O. H. Naumann, A. Pahl, C. Mardin, E. R. Tamm, and K. Brune Expression of Cyclooxygenase-1 and -2 in Normal and Glaucomatous Human Eyes Invest. Ophthalmol. Vis. Sci., October 1, 2001; 42(11): 2616 - 2624. [Abstract] [Full Text] [PDF]
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