Comparative expression profiling with kinetic RT-PCR in paired fresh frozen and archived formalin-fixed, paraffin-embedded renal cell cancer and normal tissue

Abstract

4606 Background: It would be a substantial progress if gene expression of tumor markers could be accurately analyzed on RNA isolated from formalin fixed paraffin-embedded (FFPE) tumor tissue which is routinely collected. To prove equivalence between fresh frozen and archived FFPE tissue RNA profiles, we quantified 12 different genes with kinetic RT-PCR in renal tumor and paired adjacent normal tissue archived for 8–14 years. Methods: We had access to a set of 32 clear cell renal cell cancers and its adjacent normal tissue (HELIOS Clinic, Wuppertal). Each sample existed as a FFPE tissue block and as paired fresh frozen tissue both stored over 8–14 years at RT or -80oC, respectively. RNA from FFPE tissue was isolated with a Bayer HealthCare internal silica bead-based isolation method. Each sample was analyzed with kinetic one-step RT-PCR for the gene expression of 3 housekeepers (RPL37A, GAPDH, CD63) and 9 candidate genes (EGFR, Her2/neu, Her3, Her4, EGF, TGFα, NRG1, HIF1α, VEGFα). Results: The comparative FFPE/fresh frozen expression data showed a good correlation over all data points (r = 0.87 for Ct values) and the tumor specific up- and down regulation of EGFR family genes and its ligands could be detected in both tissue types equally. We could clearly demonstrate the tumor specific up-regulation of EGFR (2-fold), TGFα (2-fold), VEGFα (4-fold) and down-regulation of EGF (60-fold), Her2/neu (4-fold), Her3 (2-fold) and Her4 (30-fold) in renal cell cancer for both tissue entities (fixed and fresh frozen). In addition a 3-dimensional Principal Component Analysis completely separated the renal tumor population from paired normal tissue in both tissue entities based on the differential gene expression. Conclusions: Here we demonstrate that a small set of genes from the EGFR family and their ligands is specifically up-/down-regulated in renal cell cancer tissue and therefore can be clearly distinguished from normal renal tissue. Furthermore, these data prove that the internal Bayer HealthCare isolation/kinetic RT-PCR detection protocol for RNA from FFPE tissue allows accurate retrospective expression profiling and validation of marker panels in archived tissue material stored for more than a decade. [Table: see text]