Global gene expression profiling of formalin-fixed paraffin-embedded tumor samples: a comparison to snap-frozen material using oligonucleotide microarrays

Abstract

Oligonucleotide microarrays are widely used to investigate gene expression in a large-scale approach. A major limitation is the dependency on frozen material to obtain high-quality ribonucleic acid because most clinical specimens are formalin-fixed and paraffin-embedded (FFPE). The ability to analyze these samples using microarrays would enlarge the investigable sample stocks manifold. We conducted a comparison of snap-frozen and FFPE tissues investigating two malignomas. Gene expression profiles were obtained from both materials of the tumors. Independently processed triplicates of snap-frozen and FFPE specimen, respectively, were two-round-amplified and hybridized on Affymetrix GeneChips (Palo Alto, CA, USA). Differentially expressed genes were identified in both FFPE and frozen material. All replicates had a correlation coefficient (R) of greater than 0.95 after normalization. Only direct comparison of FFPE to frozen replicates resulted in a mean R of 0.86, rendering a "mixed" investigation unfeasible. More than 50% (419 genes) of the more than fivefold differentially expressed genes (800 in FFPE, 685 in frozen material) were detected concomitantly regardless of the material used, which is similar to other comparisons of different gene expression analysis platforms. Thus, global gene expression analyses using solely FFPE material seem to be feasible with nearly comparable results to frozen tissue studies.

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

“…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.…”

Application of a Clinical Whole-Transcriptome Assay for Staging and Prognosis of Prostate Cancer Diagnosed in Needle Core Biopsy Specimens

“…Bibikova et al 29 and Frank et al 30 assessed the reproducibility of FFPE samples profiled with oligonucleotide arrays and found high concordance between replicate samples. Likewise, high correlations (r !…”

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.…”

“…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. 30,32,75,78 Magnitudes of differential expression are often higher in formalin-fixed paraffin-embedded tissue than in fresh frozen samples, because of the increased background noise; this increases the odds of false-positive results. 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.…”

“…30,83 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.…”