Identification of Reference Genes for Gene Expression Analysis by Real Time Quantitative PCR (RTQ-PCR) in Pancreatic Adenocarcinoma
KK Lai, X Liu. Cleveland Clinic, Cleveland, OH
Background: Pancreatic adenocarcinoma is a disease with dismal prognosis. Recent studies utilizing RTQ-PCR in patients with this cancer have identified gene expression patterns with prognostic implication. As the clinical utility of RTQ-PCR has increased, traditionally used internal reference genes have been shown to exhibit differential levels of expression in different tissues, leading to the drive to identify tissue-specific reference genes. Identification of reference genes for normalization of RTQ-PCR data from paraffin-embedded tissue samples of human pancreatic adenocarcinoma have not yet been reported.
Design: Eight eight pancreatic adenocarcinomas resected from 2000 to 2005 were included in this study. Tumor samples were either formalin fixed (n=67) or Hollandes-fixed (n=21). Total RNA was isolated from micro-dissected paraffin-embedded tumors. One-step multiplex RTQ-PCR was performed to examine expression levels of the three housekeeper genes β-actin (ACTB), hydroxymethylbilane synthase (HMBS) and ribosomal protein L13 A (RPL13A). NormFinder (v19) and geNorm (v3.5) software programs were used for stability comparisons of these 3 housekeeper genes. The programs evaluate expression stability via independent algorithms.
Results: NormFinder identified HMBS as the most stably expressed (lowest stability value, table 1), followed by RPL13A and ACTB. Fixative type did not alter the order of stability. Analysis using geNorm yielded the same order of stability, again regardless of fixative (table 2).
|Rank||Gene||Stability value||Tumor Intragroup variation (n=67)|
|Rank||Gene||Stability value||Tumor Intragroup variation (n=21)|