Abstract
Quantifying the ratio of alternatively spliced mRNA variants of genes with known alternative splicing variants is highly relevant for many applications. Herein, we describe the validation of a quantitative PCR design for the simplified quantification of known mRNA splice variants. The assay uses a single-common primer pair, dual probe design for the determination of splicing variants in a single well configuration. We used murine XBP-1 splicing variants, XBP-1S and XBP-1U, to validate and demonstrate the performance characteristics of this approach. Using synthetic XBP-1S and XBP-1U cDNA as well as cDNA synthesized from mouse beta-cell line MIN6, we established the performance parameters and dynamic range of the assay. Reliable quantification of both variants at varying concentration gradients was shown. No cross detection of XBP-1U by the XBP-1S probe was detected and only marginal XBP-1S cross detection by the XBP-1U probe was detected at high concentration gradients that are unlikely to be relevant. We demonstrated that the assay accurately detected changes of XBP-1 splice variants in mouse liver subjected to pharmacologically induced ER stress without the need for normalization to a reference gene.