Laboratory of Molecular Mechanisms of Neuronal Interactions, I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, Torez Avenue 44, 199223, St. Petersburg, Russia.
Laboratory of Molecular Mechanisms of Neuronal Interactions, I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, Torez Avenue 44, 199223, St. Petersburg, Russia.
Biochem Biophys Res Commun. 2021 Sep 10;569:174-178. doi: 10.1016/j.bbrc.2021.07.020. Epub 2021 Jul 10.
Adenosine deaminase-dependent RNA editing is a widespread universal mechanism of posttranscriptional gene function modulation. Changes in RNA editing level may contribute to various physiological and pathological processes. In the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor GluA2 subunit, A-I editing in the Q607R site leads to dramatic changes in function, making the receptor channel calcium-impermeable. A standard approach for quantifying (un)edited RNAs is based on endpoint PCR (Sanger sequencing or restriction analysis), a time-consuming and semiquantitative method. We aimed to develop RT-qPCR assays to quantify rat Q607R (A-I) edited/unedited mRNA in samples in the present work. Based on self-probing PCR detection chemistry, described initially for detecting short DNA fragments, we designed and optimised RT-qPCR assays to quantify Q607R (un)edited mRNA. We used self-probing primer PCR technology for mRNA quantification for the first time. Using a novel assay, we confirmed that Q607R GluA2 mRNA editing was increased in 14-day- (P14) or 21-day-old (P21) postnatal brain tissue (hippocampus) compared to the embryonic brain (whole brains at E20) in Wistar rats. Q607R unedited GluA2 mRNA was detectable by our assay in the cDNA of mature brain tissue compared to that derived through classical methods. Thus, self-probing primer PCR detection chemistry is an easy-to-use approach for RT-qPCR analysis of RNA editing.
腺苷脱氨酶依赖的 RNA 编辑是一种广泛存在的转录后基因功能调节的普遍机制。RNA 编辑水平的变化可能与各种生理和病理过程有关。在α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）谷氨酸受体 GluA2 亚基中，Q607R 位点的 A-I 编辑导致功能发生巨大变化，使受体通道钙离子不可渗透。一种用于定量（未）编辑 RNA 的标准方法基于终点 PCR（Sanger 测序或限制分析），这是一种耗时且半定量的方法。在本工作中，我们旨在开发用于定量大鼠 Q607R（A-I）编辑/未编辑 mRNA 的 RT-qPCR 测定法。基于最初用于检测短 DNA 片段的自我探测 PCR 检测化学，我们设计并优化了 RT-qPCR 测定法，以定量 Q607R（未）编辑 mRNA。我们首次将自我探测引物 PCR 技术用于 mRNA 定量。使用新的测定法，我们证实与胚胎脑（E20 时的整个大脑）相比，14 天（P14）或 21 天（P21）龄新生大鼠脑组织（海马体）中 Q607R GluA2 mRNA 编辑增加。与经典方法相比，我们的测定法可在成熟脑组织的 cDNA 中检测到 Q607R 未编辑的 GluA2 mRNA。因此，自我探测引物 PCR 检测化学是用于 RNA 编辑的 RT-qPCR 分析的一种简单易用的方法。
