Quantitation of TGF-beta1 mRNA in porcine mesangial cells by comparative kinetic RT/PCR: comparison with ribonuclease protection assay and in situ hybridization.
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.
Ceol
M
M
Division of Nephrology, Department of Medical and Surgical Sciences, University of Padova, Italy. giga@ux1.unipd.it
Forino
M
M
Gambaro
G
G
Sauer
U
U
Schleicher
E D
ED
D'Angelo
A
A
Anglani
F
F
D000818:Animals; D005920:Glomerular Mesangium / Q000737:chemistry*; D006493:Heparin / Q000494:pharmacology; D017403:In Situ Hybridization; D007700:Kinetics; D012333:RNA, Messenger / Q000032:analysis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases; D012680:Sensitivity and Specificity; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000235:genetics*; D053773:Transforming Growth Factor beta1
D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D006493:Heparin; D012260:Ribonucleases; D013755:Tetradecanoylphorbol Acetate