Kaneko Shohei, Yanai Katsunori, Ishii Hiroki, Aomatsu Akinori, Ito Kiyonori, Hirai Keiji, Ookawara Susumu, Ishibashi Kenichi, Morishita Yoshiyuki
Division of Nephrology, First Department of Integrated Medicine, Saitama Medical Center, Jichi Medical University;
Division of Nephrology, First Department of Integrated Medicine, Saitama Medical Center, Jichi Medical University.
J Vis Exp. 2020 Jul 8(161). doi: 10.3791/61535.
Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal failure. In recent years, the involvement of microRNAs (miRNAs) has been reported in the pathogenesis of IgA nephropathy. However, there is no established method for profiling miRNAs in IgA nephropathy using small animal models. Therefore, we developed a reliable method for analyzing miRNA in the kidney of an IgA mouse model (HIGA mouse). The goal of this protocol is to detect the altered expression levels of miRNAs in the kidneys of HIGA mice when compared with the levels in kidneys of control mice. In brief, this method consists of four steps: 1) obtaining kidney samples from HIGA mice; 2) purifying total RNA from kidney samples; 3) synthesizing complementary DNA from total RNA; and 4) quantitative reverse transcription polymerase chain reaction (qRT-PCR) of miRNAs. Using this method, we successfully detected the expression levels of several miRNAs (miR-155-5p, miR-146a-5p, and miR-21-5p) in the kidneys of HIGA mice. This new method can be applied to other studies profiling miRNAs in IgA nephropathy.
免疫球蛋白A（IgA）肾病是一种原发性肾小球肾炎，其特征是IgA异常沉积，可导致终末期肾衰竭。近年来，已有报道称微小RNA（miRNA）参与了IgA肾病的发病机制。然而，目前尚无利用小动物模型对IgA肾病中的miRNA进行分析的既定方法。因此，我们开发了一种可靠的方法来分析IgA小鼠模型（HIGA小鼠）肾脏中的miRNA。本方案的目的是检测与对照小鼠肾脏中的水平相比，HIGA小鼠肾脏中miRNA表达水平的变化。简而言之，该方法包括四个步骤：1）从HIGA小鼠获取肾脏样本；2）从肾脏样本中纯化总RNA；3）从总RNA合成互补DNA；4）对miRNA进行定量逆转录聚合酶链反应（qRT-PCR）。使用该方法，我们成功检测了HIGA小鼠肾脏中几种miRNA（miR-155-5p、miR-146a-5p和miR-21-5p）的表达水平。这种新方法可应用于其他对IgA肾病中的miRNA进行分析的研究。
