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.

RT-qPCR的需要的稳定表达的参考基因表达的精确正常化的适当的选择。然而，测试一组参考基因通常是耗时且昂贵的。在这项工作中，我们旨在开发一套多重实时PCR分析方法，用于实验室大鼠中常用管家基因的RT-qPCR分析。使用水解探针（TaqMan®）技术，我们设计并优化了三种三重qPCR检测方法（Actb + Gapdh + B2m；Rpl13a + Sdha + Ppia；Hprt1 + Pgk1 + Ywhaz）展示了最佳的PCR扩增效率（从94.7至100.5％）和可重复性。与广泛的单重分析相比，新颖的分析方法可在3个反应中对9个参考基因进行表达分析，从而有可能在Wistar大鼠的RT-qPCR实验中更省时地选择参考基因。