Related Experiment Video
Updated: Dec 20, 2025
Optimized Quantitative Assessment of Enhancer RNA Stability in Mouse Embryonic Stem Cells
Published on: November 21, 2025
Multiplex qPCR assay for assessment of reference gene expression stability in rat tissues/samples
Alexander P Schwarz1, Daria A Malygina1, Anna A Kovalenko1
1Laboratory of Molecular Mechanisms of Neural Interactions, Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 44 Prospekt Torez, 194223, Saint Petersburg, Russia.
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.
