Quantification of splice variants using molecular beacon or scorpion primers

Uncovering the relationship between the generation of alternative transcripts and cellular processes is of crucial importance in the exploration of a gene's biology. The description and quantification of the spatiotemporal splicing pattern can be one method to select the most interesting transcripts for future studies. Fluorescence-based real-time quantitative RT-PCR has recently revolutionized the possibilities for transcriptional quantification studies. In this report, Molecular Beacon and Scorpion probes have been tested as new possibilities for determining the expression level of alternative transcripts. We validated these systems by analyzing alternative splicing of exons 6, 15, and 16 of the calpain 3 gene with tissues containing large variation in the ratio of the different transcripts. We determined conditions that demonstrated that boundary probes are useful tools and good alternatives to boundary primers, when developing a system to quantify specific transcripts. We suggest that the choice of a quantification system should depend in part on the structure and base composition of the gene and may have to be determined experimentally.