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A quantitative method for comparison of expression of alternatively spliced genes using different primer pairs

Donev, Rossen Mintchev and Morgan, Bryan Paul ORCID: https://orcid.org/0000-0003-4075-7676 2006. A quantitative method for comparison of expression of alternatively spliced genes using different primer pairs. Journal of Biochemical and Biophysical Methods 66 (1-3) , pp. 23-31. 10.1016/j.jbbm.2005.11.001

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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.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Medicine
Subjects: R Medicine > R Medicine (General)
Publisher: Elsevier
ISSN: 0165-022X
Last Modified: 27 Oct 2022 08:52
URI: https://orca.cardiff.ac.uk/id/eprint/63636

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