Quantitative and selective polymerase chain reaction analysis of highly similar human alpha-class glutathione...

Alpha-class glutathione transferases (GSTs) found expressed in human tissues constitute a family of four homologous enzymes with contrasting enzyme activities. In particular, GST A3-3 has been shown to contribute to the biosynthesis of steroid hormones in human cells and is selectively expressed in steroidogenic tissues. The more ubiquitous GST A1-1, GST A2-2, and GST A4-4 appear to be primarily involved in detoxification processes and are expressed at higher levels than GST A3-3. We are interested in studying the cell and tissue expression of the GST A3-3 gene, yet the existence of highly expressed sequence-similar homologs and of several splice variants is a serious challenge for the specific detection of unique transcript species. We found that published polymerase chain reaction (PCR) primers for GST A3-3 lack the specificity required for reliable quantitative analysis. Therefore, we designed quantitative PCR (qPCR) primers with greatly increased discrimination power for the human GSTA3 full-length transcript. The improved primers allow accurate discrimination between GST A3-3 and the other alpha-class GSTs and so are of great value to studies of the expression of the GSTA3 gene. The novel primers were used to quantify GSTA3 transcripts in human embryonic liver and steroidogenic cell lines.