Quantitative expression profiling of highly degraded RNA from formalin-fixed, paraffin-embedded breast tumor biopsies by oligonucleotide microarrays

Abstract

Microarray-based gene expression profiling is well suited for parallel quantitative analysis of large numbers of RNAs, but its application to cancer biopsies, particularly formalin-fixed, paraffin-embedded (FFPE) archived tissues, is limited by the poor quality of the RNA recovered. This represents a serious drawback, as FFPE tumor tissue banks are available with clinical and prognostic annotations, which could be exploited for molecular profiling studies, provided that reliable analytical technologies are found. We applied and evaluated here a microarray-based cDNA-mediated annealing, selection, extension and ligation (DASL) assay for analysis of 502 mRNAs in highly degraded total RNA extracted from cultured cells or FFPE breast cancer (MT) biopsies. The study included quantitative and qualitative comparison of data obtained by analysis of the same RNAs with genome-wide oligonucleotide microarrays vs DASL arrays and, by DASL, before and after extensive in vitro RNA fragmentation. The DASL-based expression profiling assay applied to RNA extracted from MCF-7 cells, before or after 24 h stimulation with a mitogenic dose of 17b-estradiol, consistently allowed to detect hormone-induced gene expression changes following extensive RNA degradation in vitro. Comparable results where obtained with tumor RNA extracted from FFPE MT biopsies (6 to 19 years old). The method proved itself sensitive, reproducible and accurate, when compared to results obtained by microarray analysis of RNA extracted from snap-frozen tissue of the same tumor.

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.…”

“…Recent biotechnological advances have optimized conditions to improve the quality and quantity of RNA extracted from formalin-fixed paraffin-embedded tissues. [30][31][32] These now permit molecular analysis, including microarray-based whole-genome-wide testing, of clinically small lesions from archival tissues.…”

Gene expression of estrogen receptor, progesterone receptor and microtubule-associated protein Tau in high-risk early breast cancer: a quest for molecular predictors of treatment benefit in the context of a Hellenic Cooperative Oncology Group trial

“…Conversely, when tumoral hormone receptor mRNA was categorised as negative, immunohistochemical protein staining was evident in at least 1% of tumor cells in 37-39% of cases. This discrepancy could reflect the difficulty capturing short-lived, sensitive to degradation mRNA from FFPE tumor blocks, as well as the different fixation and turnover times of ribonucleic acids in relation to proteins [22,23]. Accumulation of the protein due to aberrant catabolism may also be a factor.…”