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.
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.
Wickert
L
L
Institute of Clinical Chemistry and Pathobiochemistry, RWTH-University Hospital Aachen, Pauwelsstr. 30, D-52074 Aachen, Germany. wickert@unicum.de
Steinkrüger
S
S
Abiaka
M
M
Bolkenius
U
U
Purps
O
O
Schnabel
C
C
Gressner
A M
AM
D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D017931:DNA Primers; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D020033:Protein Isoforms / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012015:Reference Standards; D016212:Transforming Growth Factor beta / Q000235:genetics*
D017931:DNA Primers; D020033:Protein Isoforms; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases