title: Limitations of commonly used internal controls for real-time RT-PCR analysis of renal epithelial-mesenchymal cell transition.
abstract: 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.
authors.affiliation: Department of Pediatrics, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA. gerard-elberg@ouhsc.edu
authors.forename: Gerard
authors.initials: G
authors.lastname: Elberg
authors.forename: Dorit
authors.initials: D
authors.lastname: Elberg
authors.forename: Charlotte J
authors.initials: CJ
authors.lastname: Logan
authors.forename: Lijuan
authors.initials: L
authors.lastname: Chen
authors.forename: Martin A
authors.initials: MA
authors.lastname: Turman
mesh_terms: 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
chemical_list: 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