Analysis of biological and technical variability in gene expression assays from formalin-fixed paraffin-embedded...

Formalin-fixed paraffin-embedded (FFPE) tissues are invaluable sources of biological material for research and diagnostic purposes. In this study, we aimed to identify biological and technical variability in RT-qPCR TaqMan® assays performed with FFPE-RNA from lymph nodes of classical Hodgkin lymphoma samples. An ANOVA-nested 6-level design was employed to evaluate BCL2, CASP3, IRF4, LYZ and STAT1 gene expression. The most variable genes were CASP3 (low expression) and LYZ (high expression). Total variability decreased after normalization for all genes, except by LYZ. Genes with moderate and low expression were identified and suffered more the effects of the technical manipulation than high-expression genes. Pre-amplification was shown to introduce significant technical variability, which was partially alleviated by lowering to a half the amount of input RNA. Ct and Cy0 quantification methods, based on cycle-threshold and the kinetic of amplification curves, respectively, were compared. Cy0 method resulted in higher quantification values, leading to the decrease of total variability in CASP3 and LYZ genes. The mean individual noise was 0.45 (0.31 to 0.61 SD), indicating a variation of gene expression over ~1.5 folds from one case to another. We showed that total variability in RT-qPCR from FFPE-RNA is not higher than that reported for fresh complex tissues, and identified gene-, and expression level-sources of biological and technical variability, which can allow better strategies for designing RT-qPCR assays from highly degraded and inhibited samples.