Detection and quantitative measurement of transforming growth factor-β1 (TGF-β1) gene expression using a semi-nested competitive PCR assay

A polymerase chain reaction (PCR) technique was optimized for detection and quantification of very low concentrations (down to a few molecules) of transforming growth factor beta 1 (TGF-beta 1) mRNA. The strategy involved a combination of a competitive PCR assay and a semi-nested PCR, In the present study, the semi-nested PCR technique was tested in several rat organs containing different concentrations of target mRNA, A control fragment for TGF-beta 1 was used to correct for differences in amplification of various cDNA samples, TGF-beta 1 mRNA levels were also corrected according to the abundance of the "housekeeping" gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the same samples. The differences of sensitivity among the standard (one-step) and semi-nested (two-step) competitive PCR assays for the detection of TGF-beta 1 are discussed. In conclusion, the semi-nested PCR protocol provides greatly enhanced sensitivity over standard PCR analysis. It is a reproducible and very specific method for quantification of only a few molecules of TGF-beta 1 mRNA in a background of non-target molecules. (C) 1999 Academic Press.