title: Quantification of splice variants using molecular beacon or scorpion primers.
abstract: 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.
authors.affiliation: Genethon III, CNRS URA 1923, 1, Rue de l'Internationale, Evry, 91000, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Taveau
authors.forename: Daniel
authors.initials: D
authors.lastname: Stockholm
authors.forename: Melissa
authors.initials: M
authors.lastname: Spencer
authors.forename: Isabelle
authors.initials: I
authors.lastname: Richard
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D002154:Calpain / Q000235:genetics; D017931:DNA Primers; D015342:DNA Probes; D051379:Mice; D008822:Mice, Transgenic; D009124:Muscle Proteins; D018482:Muscle, Skeletal / Q000378:metabolism; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D009124:Muscle Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D002154:Calpain; C491373:Capn3 protein, mouse