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.
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.
Mol Cell Probes. 2020 Oct;53:101611. doi: 10.1016/j.mcp.2020.101611. Epub 2020 May 30.
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.
实时 RT-qPCR 需要选择稳定表达的参照基因进行 mRNA 表达的准确归一化。然而，对参照基因进行面板测试通常既耗时又昂贵。在这项工作中，我们旨在开发一组用于实验室大鼠常用管家基因的实时 RT-qPCR 分析的多重实时 PCR 测定法。使用水解探针（TaqMan®）技术，我们设计并优化了三个三重 qPCR 测定法（Actb+Gapdh+B2m；Rpl13a+Sdha+Ppia；Hprt1+Pgk1+Ywhaz），显示出最佳的 PCR 扩增效率（从 94.7%到 100.5%）和可重复性。新测定法允许在 3 个反应中分析 9 个参照基因，与广泛使用的单重测定法相比，这使得在 Wistar 大鼠的实时 RT-qPCR 实验中更有效地选择参照基因成为可能。
