Limitations of commonly used internal controls for real-time RT-PCR analysis of renal epithelial-mesenchymal cell transition.
OBJECTIVE
Progressive renal fibrotic disease is accompanied by the massive accumulation of myofibroblasts as defined by alpha smooth muscle actin (alphaSMA) expression. We quantitated gene expression using real-time RT-PCR analysis during conversion of primary cultured human renal tubular cells (RTC) to myofibroblasts after treatment with transforming growth factor-beta1 (TGF-beta1). We report herein the limitations of commonly used reference genes for mRNA quantitation.


METHODS
We determined the expression of alphaSMA and megakaryoblastic leukemia-1 (MKL1), a transcriptional regulator of alphaSMA, 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-beta1. Thus, depending on which reference gene was used, TGF-beta1 appeared to have different effects on expression of MKL1 and alphaSMA.


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-beta1 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.
Elberg
Gerard
G
Department of Pediatrics, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA. gerard-elberg@ouhsc.edu
Elberg
Dorit
D
Logan
Charlotte J
CJ
Chen
Lijuan
L
Turman
Martin A
MA
D000199:Actins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D003199:Computer Systems / Q000592:standards; D021984:Cyclophilin A / Q000235:genetics; D005347:Fibroblasts / Q000473:pathology*; D005355:Fibrosis; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D007684:Kidney Tubules / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D020930:MAP Kinase Kinase Kinases / Q000235:genetics; D009130:Muscle, Smooth / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000473:pathology*; D011786:Quality Control; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053773:Transforming Growth Factor beta1
D000199:Actins; D012337:RNA, Ribosomal, 18S; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D020930:MAP Kinase Kinase Kinases; C433243:MAP3K9 protein, human; D021984:Cyclophilin A