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Journal article
Single well, single-common primer pair, dual probe, duplex qPCR assay for the quantification of mRNA splicing variants
Biology methods and protocols, v 6(1), 002
22 Jan 2021
PMID: 33655078
Featured in Collection : UN Sustainable Development Goals @ Drexel
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.
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Details
- Title
- Single well, single-common primer pair, dual probe, duplex qPCR assay for the quantification of mRNA splicing variants
- Creators
- Janice Wang - Northwestern UniversityWinifred P. Wong - Northwestern UniversityEmma O. Link - Drexel UniversityShantel Olivares - Northwestern UniversityCade T. Adelman - Northwestern UniversityAnne S. Henkel - Northwestern UniversityMalek El Muayed - Northwestern University
- Publication Details
- Biology methods and protocols, v 6(1), 002
- Publisher
- Oxford Univ Press
- Number of pages
- 7
- Grant note
- 5R01ES027011 / US National Institute of Health/The National Institute for Environmental Health Sciences (NIH/NIEHS)
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- College of Medicine
- Web of Science ID
- WOS:000769874000015
- Scopus ID
- 2-s2.0-85105016033
- Other Identifier
- 991021860760504721
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- Collaboration types
- Domestic collaboration
- Web of Science research areas
- Biochemical Research Methods