Quantitative monitoring of the mRNA expression pattern of the TGF-beta-isoforms (beta 1, beta 2, beta 3) during transdifferentiation of hepatic stellate cells using a newly developed real-time SYBR Green PCR

Biochem Biophys Res Commun. 2002 Jul 12;295(2):330-5. doi: 10.1016/s0006-291x(02)00669-1.


Current methods to determine the mRNA of the TGF-beta-isoforms, beta 1, beta 2, and beta 3, are not sensitive enough to detect small alterations in the expression levels. Therefore, we established a SYBR Green I-based real-time quantitative PCR procedure with fragment-specific standards. The advantage of gene-specific quantification is the possibility to be abstain from the need to compare results with a house-keeping gene having a different sequence and PCR efficiency. Reproducibility of the results and analytical variances of the real-time PCR assays were tested. In transdifferentiating rat hepatic stellate cells (HSC) the TGF-beta 1-mRNA was found to be the predominant isoform expressed followed by TGF-beta 3 and low amounts of TGF-beta 2-mRNA. An alteration of the TGF-beta 1,-beta 2, and -beta 3 ratio during HSC transdifferentiation could not be detected. Furthermore, the GAPDH mRNA expression varied during HSC activation, and thus is not recommended as a standard in real-time PCR quantifications.

MeSH terms

  • Animals
  • Base Sequence
  • Cell Differentiation*
  • DNA Primers
  • Glyceraldehyde-3-Phosphate Dehydrogenases / genetics
  • Hepatocytes / cytology
  • Hepatocytes / metabolism*
  • Male
  • Polymerase Chain Reaction / methods*
  • Protein Isoforms / genetics*
  • RNA, Messenger / genetics*
  • Rats
  • Rats, Sprague-Dawley
  • Reference Standards
  • Transforming Growth Factor beta / genetics*


  • DNA Primers
  • Protein Isoforms
  • RNA, Messenger
  • Transforming Growth Factor beta
  • Glyceraldehyde-3-Phosphate Dehydrogenases