Quantitation of TGF-beta 1 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 ribonuc lease protection assay (RPA), in situ hybridisation (ISH), and quantitative reverse transcription-polymerase chain reaction (RTIPCR). These methods di ffer considerably in their sensitivity and precision in detecting and quant ifying low abundance mRNA. Although there is evidence that RTIPCR can be pe rformed in a quantitative manner, the quantitative capacity of this method is generally underestimated. To demonstrate that the comparative kinetic RT IPCR strategy - which uses a housekeeping gene as internal standard - is a quantitative method to detect significant differences in mRNA levels betwee n different samples, the inhibitory effect of heparin on phorbol 12-myrista te 19-acetate( PMA)-induced-TGF-beta1 m RNA expression was evaluated by RT/ PCR and RPA, the standard method of mRNA quantification, and the results we re compared. The reproducibility of RT/PCR amplification was calculated by comparing the quantity of G3PDH and TGF-beta1 PCR products, generated durin g the exponential phases, estimated from two different RT/PCR (GBPDH, r = 0 .968, P = 0.0000; TGF-beta1, r = 0.966, P = 0.0000). The quantitative capac ity of comparative kinetic RT/PCR was demonstrated by comparing the results obtained from RPA and RT/PCR using linear regression analysis. Starting fr om the same RNA extraction, but using only 1 % of the RNA for the RTIPCR co mpared to RPA, significant correlation was observed (r = 0.984, P = 0.0004) . Moreover the morphometric analysis of]SH signal was applied for the semi- quantitative evaluation of the expression and localisation of TGF-beta1 mRN A in the entire cell population. Our results demonstrate the close similari ty of the RT/PCR and RPA methods in giving quantitative information on mRNA expression and indicate the possibility to adopt the comparative kinetic R T/PCR as reliable quantitative method of mRNA analysis. (C) 2001 Wiley-Liss . Inc.