Preliminary Comparison of Quantity, Quality, and Microarray Performance of RNA Extracted From Formalin-fixed, Paraffin-embedded, and Unfixed Frozen Tissue Samples

Abstract

S U M M A R Y Microarrays have been used to simultaneously monitor the expression of thousands of genes from biological samples, an approach that can potentially uncover previously unrecognized functions of genes. Microarray analyses can rarely be conducted retrospectively because of the requirement for RNA to be obtained from fresh or unfixed frozen tissues. Archived pathology specimens would need to be used for retrospective analyses, and these are typically preserved as formalin-fixed, paraffin-embedded (FFPE) tissue. Formalin-fixed tissues have been shown to yield compromised RNA compared with that obtained from frozen tissue. To begin to assess the performance of RNA extracted from FFPE samples on a microarray format, we compared RNA from a model system of pelleted lipopolysaccharidestimulated human bone marrow stromal cells that were snap frozen with RNA from FFPE cells. RNA integrity and Affymetrix quality control parameters were assessed, and differentially regulated genes were analyzed with Ingenuity Pathway Analysis software. Results demonstrate that both snap-frozen and FFPE samples yielded intact RNA suitable for amplification prior to Affymetrix GeneChip analysis. Although some transcriptional information was lost with RNA extracted from the FFPE samples, Ingenuity Pathway Analysis revealed that the major pathways identified as affected by drug treatment were similar. Results show that FFPE samples are amenable to Affymetrix GeneChip analysis, expanding the possibility for expression profiling on archived tissue blocks in pathology laboratories.

Usefulness and efficiency of formalin-fixed paraffin-embedded specimens from laryngeal squamous cell carcinoma in HPV detection by IHC and PCR/DEIA.

“…We demonstrated that FFPE tissue has high sensitivity, specificity and accuracy in comparison with deep-frozen tissue for HPV detection in LSCC. These findings confirm and expand the results of studies of other authors for lung and cervical cancers [16][17][18]. Farragher et al [16] observed a remarkably high level of present cells in both frozen and FFPE samples.…”

Acquisition of biologically relevant gene expression data by Affymetrix microarray analysis of archival formalin-fixed paraffin-embedded tumours

“…It is nevertheless desirable to improve the sensitivity of FFPET arrays. Low sensitivity rates of around 50% relative to FT arrays have been reported by others (Bibikova et al, 2004;Scicchitano et al, 2006) and may be due to the presence of residual nonreversed chemical modifications that reduce the efficiency or completely prevent in vitro transcription to cRNA (Godfrey et al, 2000;Bibikova et al, 2004;Lee et al, 2005b). Our archival FFPET RNA samples were similarly (and extensively) degraded, yet cRNA yields varied widely (5 -28 mg).…”

“…Whereas RT -PCR interrogation of archival FFPET has proven to be a reliable tool and is widely used (Cronin et al, 2004), this approach is unsuitable for large scale discovery-based investigation owing to the limited number of genes that can be assayed in one experiment. RNA from freshly prepared FFPET may be minimally degraded (von Ahlfen et al, 2007) and their transcript profiles correlate very well with those from paired unfixed FT (Lee et al, 2005a;Scicchitano et al, 2006;Frank et al, 2007), but these samples lack the requisite clinical follow-up data for correlation with expression data.…”

Differential gene expression profiles of neurothekeomas and nerve sheath myxomas by microarray analysis

“…These advances include newer formalin-fixed paraffin-embedded tissue extraction protocols, cDNA-mediated annealing, selection, extension and ligation, the addition of random hexamer priming to oligo(dT) priming and newly developed array platforms with redesigned probes. [30][31][32][33][34]57,[67][68][69][70][71][72][73][74][75][76][77][78][79][80][81][82] Although sensitivity is low, high specificity and positive predictive value suggest that transcript detection is reliable from formalin-fixed paraffin-embedded tissue. 33 Although RNA isolation techniques from formalin-fixed paraffin-embedded tissues have improved, problems remain, largely because of the greater fragmented nature of RNA in formalin-fixed paraffin-embedded tissue.…”

“…30,83 Despite these concerns, several studies have shown comparable biological information between genome-wide microarray analysis from matched fresh frozen and formalin-fixed paraffin-embedded tissue. 30,32,72,74,75,77,78 The use of formalin-fixed paraffin-embedded rather than fresh frozen tissue clearly allows more practical applications of gene microarray analysis. The National Cancer Institute estimates that 85% of all cancer surgeries are performed in community hospitals, which historically have not had the resources for procuring snap frozen tumor samples.…”