Expression profiling of archival renal tumors by quantitative PCR to validate prognostic markers

Formalin-fixed paraffin-embedded (FFPE) tissues are routinely stored by most pathology departments and are a widely available resource for discovery of clinically useful biomarkers. We describe our method for optimizing quantitative reverse transcription PCR (RT-PCR) for expression analysis using frozen and archival tissue. Commonly used reference genes were evaluated for stability of expression in normal kidney and clear cell renal cell carcinoma (RCC). Optimal reference genes for calculating normalization factors for RT-PCR were ACTB, RPL13A, GUS, RPLP0, HPRT1, and SDHA when using FFPE RCC. The optimal reference genes when using frozen RCC were ACTB, RPL13A, and GUS, confirming that use of multiple reference genes improves accuracy when intact RNA from frozen renal tumors are used. Expression of 16 markers previously reported to have prognostic significance in RCC was determined in 23 matching frozen and FFPE renal tumors, representing a range of tumor grades and stages; correlation coefficient for expression measured in frozen and FFPE tumors was 0.921 (P < 0.001). All markers predicted survival when frozen tumors were used and 14 of the 16 markers predicted survival when FFPE tumors were used as the source of RNA. An optimized RT-PCR assay can accurately measure expression of most prognostic tumor markers.