Multiplex qPCR assay for assessment of reference gene expression stability in rat tissues/samples

Alexander P Schwarz; Daria A Malygina; Anna A Kovalenko; Alexander N Trofimov; Aleksey V Zaitsev

doi:10.1016/j.mcp.2020.101611

Multiplex qPCR assay for assessment of reference gene expression stability in rat tissues/samples

Mol Cell Probes. 2020 Oct:53:101611. doi: 10.1016/j.mcp.2020.101611. Epub 2020 May 30.

Authors

Alexander P Schwarz¹, Daria A Malygina², Anna A Kovalenko², Alexander N Trofimov³, Aleksey V Zaitsev²

Affiliations

¹ Laboratory of Molecular Mechanisms of Neural Interactions, Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 44 Prospekt Torez, 194223, Saint Petersburg, Russia. Electronic address: aleksandr.pavlovich.schwarz@gmail.com.
² Laboratory of Molecular Mechanisms of Neural Interactions, Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 44 Prospekt Torez, 194223, Saint Petersburg, Russia.
³ Laboratory of Neurobiology of the Brain Integrative Functions, I. P. Pavlov Department of Physiology, Institute of Experimental Medicine (IEM), 12 Acad. Pavlov st, 197376, Saint Petersburg, Russia.

PMID: 32485234
DOI: 10.1016/j.mcp.2020.101611

Abstract

RT-qPCR requires an adequate choice of stably expressed reference genes for accurate normalization of mRNA expression. However, testing a panel of reference genes is often time-consuming and expensive. In this work, we aimed to develop a set of multiplex real-time PCR assays for RT-qPCR analysis of commonly used housekeeping genes in laboratory rats. Using Hydrolysis probe (TaqMan®) technology, we have designed and optimized three triplex qPCR assays (Actb + Gapdh + B2m; Rpl13a + Sdha + Ppia; Hprt1+Pgk1+Ywhaz) demonstrating optimal PCR amplification efficiencies (from 94.7 to 100.5%) and repeatability. Novel assays allow expression analysis of 9 reference genes in 3 reactions making possible a more time-efficient choice of reference genes in RT-qPCR experiments in Wistar rats in comparison with widespread singleplex assays.

Keywords: Gene expression analysis; Multiplex real-time PCR assay; RT-qPCR; Rat; Reference gene stability.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Line
Gene Expression Profiling / standards*
Genes, Essential*
Multiplex Polymerase Chain Reaction / standards*
Rats
Rats, Wistar
Real-Time Polymerase Chain Reaction / standards
Reference Standards