Selection of REFERENCE genes for normalization of qRT-PCR data derived from FFPE breast tumors.

Gene expression analysis of formalin-fixed paraffin-embedded (FFPE) material by quantitative real-time polymerase chain reaction is a valuable tool for the retrospective analysis of clinical samples. The degraded nature of RNA from FFPE tissue can lead to variation in detection of gene expression between samples, hence, it is necessary to select reference genes that can reflect this variation. To identify suitable references for the normalization of target genes in FFPE breast tumors, expression of 10 potential references were measured in 10 endocrine therapy-naive, primary invasive breast tumors. The most stably expressed reference genes were identified with the use of the freely available algorithm geNorm. Using the geometric mean of MRPL19, TBP, and TFRC as a reference factor for FFPE tissue, there was a good correlation of ERalpha and HER2 measurements between FFPE and matched frozen tumors, indicating that this combination of reference genes successfully removed the effect of RNA degradation on quantitative real-time polymerase chain reaction analysis.