Limitations of commonly used internal controls for real-time RT-PCR analysis of renal epithelial-mesenchymal cell transition

Background/Aims: Progressive renal fibrotic disease is accompanied by the massive accumulation of myofibroblasts as defined by alpha smooth muscle actin (alpha SMA) expression. We quantitated gene expression using realtime RT-PCR analysis during conversion of primary cultured human renal tubular cells (RTC) to myofibroblasts after treatment with transforming growth factor beta(1) (TGF-beta 1). We report herein the limitations of commonly used reference genes for mRNA quantitation. Methods: We determined the expression of alpha SMA and megakaryoblastic leukemia-1 (MKL1), a transcriptional regulator of alpha SMA, by quantitative real-time PCR using three common internal controls, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A and 18S rRNA. Results: Expression of GAPDH mRNA and cyclophilin A mRNA, and to a lesser extent, 18S rRNA levels varied over time in culture and with exposure to TGF-beta 1. Thus, depending on which reference gene was used, TGF-beta 1 appeared to have different effects on expression of MKL1 and alpha SMA. Conclusions: RTC converting to myofibroblasts in primary culture is a valuable system to study renal fibrosis in humans. However, variability in expression of reference genes with TGF-beta(1) treatment illustrates the need to validate mRNA quantitation with multiple reference genes to provide accurate interpretation of fibrosis studies in the absence of a universal internal standard for mRNA expression. Copyright (c) 2006 S. Karger AG, Basel.