Quantitation of TGF-beta1 mRNA in porcine mesangial cells by comparative kinetic RT/PCR: comparison with ribonuclease protection assay and in situ hybridization

J Clin Lab Anal. 2001;15(4):215-22. doi: 10.1002/jcla.1030.


Gene expression can be examined with different techniques including ribonuclease protection assay (RPA), in situ hybridisation (ISH), and quantitative reverse transcription-polymerase chain reaction (RT/PCR). These methods differ considerably in their sensitivity and precision in detecting and quantifying low abundance mRNA. Although there is evidence that RT/PCR can be performed in a quantitative manner, the quantitative capacity of this method is generally underestimated. To demonstrate that the comparative kinetic RT/PCR strategy-which uses a housekeeping gene as internal standard-is a quantitative method to detect significant differences in mRNA levels between different samples, the inhibitory effect of heparin on phorbol 12-myristate 13-acetate (PMA)-induced-TGF-beta1 mRNA expression was evaluated by RT/PCR and RPA, the standard method of mRNA quantification, and the results were compared. The reproducibility of RT/PCR amplification was calculated by comparing the quantity of G3PDH and TGF-beta1 PCR products, generated during the exponential phases, estimated from two different RT/PCR (G3PDH, r = 0.968, P = 0.0000; TGF-beta1, r = 0.966, P = 0.0000). The quantitative capacity of comparative kinetic RT/PCR was demonstrated by comparing the results obtained from RPA and RT/PCR using linear regression analysis. Starting from the same RNA extraction, but using only 1% of the RNA for the RT/PCR compared to RPA, significant correlation was observed (r = 0.984, P = 0.0004). Moreover the morphometric analysis of ISH signal was applied for the semi-quantitative evaluation of the expression and localisation of TGF-beta1 mRNA in the entire cell population. Our results demonstrate the close similarity of the RT/PCR and RPA methods in giving quantitative information on mRNA expression and indicate the possibility to adopt the comparative kinetic RT/PCR as reliable quantitative method of mRNA analysis.

Publication types

  • Comparative Study

MeSH terms

  • Animals
  • Glomerular Mesangium / chemistry*
  • Heparin / pharmacology
  • In Situ Hybridization
  • Kinetics
  • RNA, Messenger / analysis*
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction*
  • Ribonucleases
  • Sensitivity and Specificity
  • Swine
  • Tetradecanoylphorbol Acetate / pharmacology
  • Transforming Growth Factor beta / genetics*
  • Transforming Growth Factor beta1


  • RNA, Messenger
  • Transforming Growth Factor beta
  • Transforming Growth Factor beta1
  • Heparin
  • Ribonucleases
  • Tetradecanoylphorbol Acetate