SEMIQUANTITATIVE MEASUREMENT OF GENE-EXPRESSION BY RT-PCR - A CAUTIONARY TALE

The polymerase chain reaction (PCR) theoretically offers a very powerf ul means of quantifying gene expression in cells and tissues of differ ent histological classes. From a technical viewpoint however, the use of RT-PCR to quantify gene expression can be demanding with poor repro ducibility arising from several diverse sources. In this study, we des cribe and fully characterise an RT-PCR assay which generates highly re producible estimates of DNA polymerase beta gene expression relative t o the expression of beta-actin in a semi-quantitative manner. Particul ar emphasis has been placed on the efficiency of first strand cDNA syn thesis and to aspects of primer design. In addition, various aspects a ssociated with the quantification of gene expression required to gener ate reproducible results are discussed. Using the techniques described herein, the quantification of DNA polymerase beta expression in three independent experiments using human colon carcinoma cells was highly reproducible with ratios of target to internal standard gene expressio n of 3.68x10(-4) +/- 0.23x10(-4). Provided that careful consideration is given to key areas of the RT-PCR assay during experimental work up procedures, this assay can be used to provide an accurate measure of g ene expression in cell lines.