Identification of methods for use of formalin-fixed, paraffin-embedded tissue samples in RNA expression profiling

Abstract

Formalin-fixed paraffin-embedded (FFPE) tissue samples are a potentially valuable resource of expression information for medical research, but are under-utilized due to degradation and modification of the RNA. Using a random primer-based RNA amplification strategy, we have evaluated multiple protocols for the extraction and isolation of RNA from FFPE samples. We found that the RecoverAll RNA isolation procedure with three or four slices (ten-microns in thickness), supplemented with additional DNAse, gave optimal results. RNA integrity as assessed by Agilent Bioanalyzer, and amplification of the 28S ribosomal RNA, were predictive for the number of genes detected on Affymetrix arrays. We obtained expression data for colon and lung tumor and normal FFPE samples and matched frozen samples and found a high correlation between frozen and matched FFPE samples (R(2) between 0.82 and 0.89), while the signature sets in tumor versus normal comparisons were also quite similar. QPCR confirmed all 16 of the differential expression results from the microarrays that we tested. Differentially expressed signature genes from tumor versus matched normal FFPE tissue from colon and lung were identified as cancer-related, with 95 colon tumor and 67 lung tumor genes identified, respectively.

Heterogeneity in signaling pathways of gastroenteropancreatic neuroendocrine tumors: a critical look at notch signaling pathway

“…Our results showed an almost perfect correlation (R 2 ¼ 0.97) between the microarray and qRT-PCR data. Together with other reports, 8,20 our results illustrated that formalinfixed and paraffin-embedded tissue material is valuable in evaluating the molecular pathology of neuroendocrine tumors-a particularly useful resource when fresh-frozen tissue is scarce. However, our data also suggest limited sensitivity of the formalin-fixed and paraffin-embedded tissue profiling approach.…”

“…12 However, microarray analysis using formalin-fixed and paraffinembedded tissue is a new and evolving field. 8,20 The technical challenges of formalin-fixed and paraffin-embedded tissue profiling are significant, formalin fixation leads to degradation and chemical modification of the RNA with extensive cross-links between various biomolecules in the tissue, thus, hindering analysis. 21 However, significant improvements have been made in RNA isolation and amplification-a series of reports have suggested that gene profiling on formalin-fixed and paraffinembedded tissue material is now feasible.…”

“…This traditional view has recently been challenged, and several groups have reported quantitative gene expression and microarray profiling data from formalin-fixed and paraffin-embedded tissue blocks. 7,8 The goal of this study was to explore signal transduction pathways and gene expression profile in three common gastrointestinal neuroendocrine tumors: rectum, small intestine and pancreas, and assess the heterogeneity in the signaling pathways across these tumors. Identification of novel signaling pathway may uncover potential druggable targets.…”

Analysis of mRNA Expression Levels in FFPE Testicular Biopsies Using Real-time PCR Arrays

“…The Ct threshold was set to an arbitrary value of 0.067761 after averaging all plates and performing manual inspection for best-fit, while the baseline was set to 3 cycles below the lowest Ct. Afterwards, the Ct values for each sample were exported into separate Microsoft Excel files for webbased RT 2 Profiler PCR Array Data Analysis 3.5 (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php). Quantification of the relative changes of gene expression levels (fold-change) was performed using the 2 −ΔΔCt method.…”

“…However, the isolation and downstream analysis of nucleic acids from these samples are technically difficult due to chemical modifications and degradation associated with the fixation process and storage. 2 In particular, the presence of formaldehyde induces crosslinking between nucleic acids and proteins, and the low pH (<1) induces fragmentation. 3,4 Nevertheless, FFPE-archived tissues are more easily accessible than fresh or frozen tissues, thus much effort has been made to use FFPE tissues for different genetic analysis.…”