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Comparative Study
.2001 Feb;158(2):419-29.
doi: 10.1016/S0002-9440(10)63985-5.

Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue

Affiliations
Comparative Study

Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue

K Specht et al. Am J Pathol.2001 Feb.

Abstract

Formalin-fixed, paraffin-embedded tissue is the most widely available material for retrospective clinical studies. In combination with the potential of genomics, these tissues represent an invaluable resource for the elucidation of disease mechanisms and validation of differentially expressed genes as novel therapeutic targets or prognostic indicators. We describe here an approach that, in combination with laser-assisted microdissection allows quantitative gene expression analysis in formalin-fixed, paraffin-embedded archival tissue. Using an optimized RNA microscale extraction procedure in conjunction with real-time quantitative reverse transcriptase-polymerase chain reaction based on fluorogenic TaqMan methodology, we analyzed the expression of a panel of cancer-relevant genes, EGF-R, HER-2/neu, FGF-R4, p21/WAF1/Cip1, MDM2, and HPRT and PGK as controls. We demonstrate that expression level determinations from formalin-fixed, paraffin-embedded tissues are accurate and reproducible. Measurements were comparable to those obtained with matching fresh-frozen tissue and neither fixation grade nor time significantly affected the results. Laser microdissection studies with 5-microm thick sections and defined numbers of tumor cells demonstrated that reproducible quantitation of specific mRNAs can be achieved with only 50 cells. We applied our approach to HER-2/neu quantitative gene expression analysis in 54 microdissected tumor and nonneoplastic archival samples from patients with Barrett's esophageal adenocarcinoma and showed that the results matched those obtained in parallel by fluorescence in situ hybridization and immunohistochemistry. Thus, the combination of laser-assisted microdissection and real-time TaqMan reverse transcriptase-polymerase chain reaction opens new avenues for the investigation and clinical validation of gene expression changes in archival tissue specimens.

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Figures

Figure 1.
Figure 1.
Quantitative gene expression analysis ofEGF-R mRNA measured by real-time TaqMan QRT-PCR in matching frozen and formalin-fixed, paraffin-embedded HT29 human tumor xenografts.A: Five-μm section of a formalin-fixed, paraffin-embedded HT29 tumor xenograft stained with H&E, demonstrating the homogenous tumor histology. Original magnification, ×400.B: Serial dilutions of total RNA extracted from matching frozen and formalin-fixed, paraffin-embedded tissue samples from a HT29 tumor xenograft were subjected to 45 cycles of real-time TaqMan QRT-PCR of theEGF-R sequence. Shown are the PCR products on a 3% agarose gel after electrophoresis and ethidium bromide staining.Lane M:MSP I-digested pUC molecular weight DNA.Lanes 1 and7: 50 ng RNA.Lanes 2 and8: 10 ng RNA.Lanes 3 and9: 2 ng RNA.Lanes 4 and10: 0.4 ng RNA.Lanes 5 and11: 0.08 ng RNA.Lanes 6 and12: 16 pg RNA. No signals were generated using no-template control reactions (Lane 13) or genomic DNA (Lane 14). Thearrow indicates the 93-bpEGF-R amplification product.C: The logarithm of the input RNA amount of the same samples is plottedversus the threshold cycle (Ct) monitored during real-time TaqMan QRT-PCR. Amplification efficiency of theEGF-R gene in matching frozen and formalin-fixed, paraffin-embedded tissue samples from HT29 tumor xenografts is comparable, as indicated by similar slopes of the regression lines. All points represent the mean of duplicate PCR amplifications, but error bars are too small to be visible.
Figure 2.
Figure 2.
Quantitative determination of gene expression measured by real-time TaqMan QRT-PCR in matching frozen and formalin-fixed, paraffin-embedded HT29 and A431 human tumor xenografts. Levels ofEGF-R,HER-2/neu,FGF-R4,p21/WAF1/Cip1,MDM2, andPGK mRNAs were determined by QRT-PCR and all measurements are shown relative to the expression levels of thePGK housekeeping gene. Results shown are the mean of three independent RNA isolations from single consecutive 5-μm sections ±SEM (n = 3).
Figure 3.
Figure 3.
Effect of amplicon size on quantitative gene expression analysis by TaqMan QRT-PCR in matched frozen and formalin-fixed, paraffin-embedded tissue samples from A431 xenograft tumors. Seven primer/probe pairs for QRT-PCR were tested that amplify 66-, 98-, 122-, 158-, 182-, 319-, and 374-base-long portions ofp21/WAF1/Cip1 mRNA. The same forward primer and TaqMan probe were used in each case.A: Absolute Ct values for the seven primer pair combinations. Best results are obtained in FFPE tissue with QRT-PCR primer pairs spanning fragments <100 bp. Shown are the results of duplicate measurements but the standard deviations are too small to be seen.B: PCR products on a 3% agarose gel after electrophoresis and ethidium bromide staining.Lane M: Molecular Weight Marker VIII (Roche Molecular Biochemicals). No PCR product is generated from FFPE A431 tumor xenograft tissue with fragments >374 bp.
Figure 4.
Figure 4.
Influence of the formalin fixation procedure on quantitative gene expression analysis by TaqMan QRT-PCR. Liver and uterus with leiomyoma tissue samples that had been fixed for 20 hours were cut in sequential sections of 1 cm to a depth of 3 cm (liver) or 6 cm (uterus with leiomyoma). Quantitation ofEGF-R,HER-2/neu,FGF-R4,p21/WAF1/Cip1,MDM2, andHPRT (liver) andp21/WAF1/Cip1andHPRT (uterus and leiomyoma) was determined relative toPGK as a housekeeping gene in single 5-μm sections of the sequential blocks. Change in gene expression of the six different genes is shown relative to a 5-μm section at 1-cm depth for liver (A) and uterus with myometrium and leiomyoma (B). Shown are the results of two different measurements, each run in duplicate ±SEM. (C) Absolute Ct values for PGK and HPRT that were used for quantitation of relative expression of the mRNAs.
Figure 5.
Figure 5.
Specificity of the TaqMan QRT-PCR amplification.A: Real-time RT-PCR amplification plot ofTBP mRNA measured in formalin-fixed, paraffin-embedded liver, myometrium and leiomyoma tissues, and a prostate cancer specimen. mRNA-specific signals are detectable in all four tissue types.B: Real-time RT-PCR amplification plot ofPSA mRNA in the same tissues as indicated above, showing the specific detection of the PSA mRNA transcript only in the prostate cancer specimen.C: The same PCR products subjected to 3% agarose gel electrophoresis and ethidium bromide staining.Lane M: Molecular Weight Marker VIII (Roche Molecular Biochemicals). Thearrow indicates the 110-bp fragment of the Molecular Weight Marker VIII.
Figure 6.
Figure 6.
Quantitative gene expression analysis by TaqMan QRT-PCR after laser-assisted microdissection from formalin-fixed, paraffin-embedded tissues samples. Relative fold change in gene expression in microdissected A431 and HT29 tumor xenografts was calculated relative to gene expression in a complete 5-μm section. Defined numbers of cells (ncell = ∼10000, ∼1000, 100, and 50) were microdissected from 5-μm H&E-stained formalin-fixed, paraffin-embedded sections, followed by quantitation ofEGF-R,HER-2/neu,FGF-R4,p21/WAF1/Cip1,MDM2, andPGK mRNAs by real-time RT-PCR amplification. Expression of the different transcripts was examined from the same reverse transcription reaction and were determined relative toPGK as a housekeeping gene. Shown are the results of three independent cell picking, RNA isolation, and QRT-PCR amplifications ±SEM relative to a complete 5-μm section.
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