Quantitation of mRNA by Competitive RT-PCR and Silver Staining of Polyacrylamide Gels

We present here the development of a rapid, simple and reliable nonradioactive method for the quantification of mRNA by competitive reverse transcription polymerase chain reaction (cRT-PCR) and silver staining after the polyacrylamide gel electrophoresis (PAGE). This technique does not require any labeling or blotting procedures. In this method, the RNA is reverse transcribed in the presence of an internal cellular RNA standard (cRNA) and amplified by the PCR using specific set of primers. The silver staining and scanning densitometry of the polyacrylamide gel allows the detection and quantitation of the target sequence. The quantitation of the target mRNA is performed by comparison with the cRNA and expressed per g of total cellular RNA (TC-RNA). This technique was used to study the platelet derived growth factor-A (PDGF-A) mRNA levels in the peripheral blood mononuclear cells (PBMN) from adult healthy human subjects (n = 10) as a measure of gene expression. We demostrate that use of cRT-PCR and silver staining provides sensitive and reproducible quantitation linear with the amount of PDGF-A mRNA in the sample. This simple method can be adapted to study the expression of any cellular or viral gene of known sequence and no radioactive substance or blotting method is required to quantify PCR products.