title: A quantitative method for comparison of expression of alternatively spliced genes using different primer pairs.
abstract: In this paper we describe a novel two-step method for comparison of expression of alternatively spliced genes by quantitative PCR (QPCR) applying different primer pairs. As a model system we used rat decay accelerating factor (DAF; CD55) mRNA, which comprises three different isoforms: soluble (sDAF), transmembrane (tmDAF) and glycosyl-phosphatidylinositol (GPI) anchored (gpiDAF) forms. The first step was to prepare solid phase specific for each mRNA isoform and purify the three DAF-forms from total RNA. We then assessed amplification efficiency of primer pairs designed to recognise each of the isoforms using equimolar amounts of the three purified DAF mRNAs. The final step in our assay was to compare expression of the three DAF-isoforms in testis by QPCR taking into account the efficiency of their amplification to enable quantification. The RNA capture/QPCR method we described here can be used for quantifying the expression ratios of alternatively spliced mRNAs from a single gene or for direct comparison of expression of different genes.
authors.affiliation: Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK.
authors.forename: Rossen M
authors.initials: RM
authors.lastname: Donev
authors.forename: B Paul
authors.initials: BP
authors.lastname: Morgan
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000032:analysis* / Q000235:genetics*; D004274:DNA, Recombinant / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000235:genetics; D051381:Rats
chemical_list: D017931:DNA Primers; D004274:DNA, Recombinant; D020033:Protein Isoforms