Quantifying 35 transcripts in a single tube: Model-based calibration of the GeXP RT-PCR assay

Quantitative analysis of differential gene expression is of central importance in molecular life sciences. The Gene eXpression Profiling technology (GeXP) relies on multiplex RT-PCR and subsequent capillary electrophoretic separation of the amplification products and allows to quantify the transcripts of up to approximately 35 genes with a single reaction and one dye. Here, we provide a kinetic model of primer binding and PCR product formation as the rational basis for taking and evaluating calibration curves. With the help of a purposeful designed data processing workflow supported by easy-to-use Perl scripts for calibration, data evaluation, and quality control, the calibration procedure and the model predictions were confirmed and the robustness and linearity of transcript quantification demonstrated for differentiating Physarum polycephalum plasmodial cells. We conclude that GeXP analysis is a robust, sensitive, and useful method when the transcripts of tens to few hundred genes are to be precisely quantified in a high number of samples.