Single well, single-common primer pair, dual probe, duplex qPCR assay for the quantification of mRNA splicing variants
| Autor: | Winifred P.S. Wong, Anne S. Henkel, Janice Wang, Cade T Adelman, Malek El Muayed, Shantel Olivares, Emma O. Link |
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| Rok vydání: | 2021 |
| Předmět: |
Messenger RNA
XBP-1 Chemistry Alternative splicing Computational biology General Biochemistry Genetics and Molecular Biology splice variant quantification qPCR common primer pair Real-time polymerase chain reaction Complementary DNA RNA splicing AcademicSubjects/SCI00960 PCR probe splice Innovations duplex qPCR Primer (molecular biology) General Agricultural and Biological Sciences single primer pair Gene |
| Zdroj: | Biology Methods & Protocols |
| ISSN: | 2396-8923 |
| DOI: | 10.1093/biomethods/bpab002 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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