Single well, single-common primer pair, dual probe, duplex qPCR assay for the quantification of mRNA splicing variants

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...

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Bibliographic Details
Published in:	Biology methods and protocols Vol. 6; no. 1; p. bpab002
Main Authors:	Wang, Janice, Wong, Winifred P, Link, Emma O, Olivares, Shantel, Adelman, Cade T, Henkel, Anne S, El Muayed, Malek
Format:	Journal Article
Language:	English
Published:	England Oxford University Press 2021
Subjects:	Innovations splice variant quantification PCR probe single primer pair duplex qPCR qPCR common primer pair XBP-1
Online Access:	Get full text
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Janice Wang and Winifred P. Wong contributed equally to this work.
ISSN:	2396-8923 2396-8923
DOI:	10.1093/biomethods/bpab002