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

Janice Wang, Winifred P. Wong, Emma O. Link, Shantel Olivares, Cade T. Adelman, Anne S. Henkel, Malek El Muayed*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Article numberbpab002
JournalBiology Methods and Protocols
Volume6
Issue number1
DOIs
StatePublished - 2021

Keywords

  • PCR probe
  • XBP-1
  • common primer pair
  • duplex qPCR
  • qPCR
  • single primer pair
  • splice variant quantification

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

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