Quantitative monitoring of the mRNA expression pattern of the TGF-β-isoforms (β1, β2, β3) during transdifferentiation of hepatic stellate cells using a newly developed real-time SYBR Green PCR

Current methods to determine the mRNA of the TGF-beta-isoforms, beta1, beta2 and beta3, 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-beta1-mRNA was found to be the predominant isoform expressed followed by TGF-beta3 and low amounts of TGF-beta2-mRNA. Ail alteration of the TGF-beta1,-beta2, and -beta3 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. (C) 2002 Elsevier Science (USA). All rights reserved.