检测免疫球蛋白A肾病小鼠肾脏中的microRNA表达
Summary
microRNA参与IgA肾病的发病机制。我们已经开发出一种可靠的方法来检测IgA肾病小鼠模型（HIGA小鼠）肾脏中的microRNA表达水平。这种新方法将有助于检查miRNA是否参与IgA肾病。
Abstract
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沉积异常，导致终末期肾衰竭。近年来，在IgA肾病的发病机制中报道了microRNA（miRNA）的参与。然而，没有使用小动物模型分析IgA肾病中miRNA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。该协议的目标是检测与对照小鼠肾脏中的水平相比，HIGA小鼠肾脏中miRNA的表达水平改变。简而言之，该方法包括四个步骤：1）从HIGA小鼠获得肾脏样本;2）从肾脏样本中纯化总RNA;3）从总RNA合成互补DNA;4）miRNA的定量逆转录聚合酶链反应（qRT-PCR）。使用这种方法，我们成功地检测了HIGA小鼠肾脏中几种miRNA（miR-155-5p，miR-146a-5p和miR-21-5p）的表达水平。这种新方法可以应用于其他分析IgA肾病中miRNA的研究。
Introduction
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在肾小球系膜区域1,2中的异常沉积。它是最常见的原发性肾小球肾炎，在20%-40%的患者中导致终末期肾衰竭2。病因尚不清楚，但与持续性黏膜感染有关1,3。皮质类固醇、免疫抑制剂和肾素-血管紧张素系统抑制剂已被提出作为治疗方法1,3，但尚未完全确立3。因此，需要进一步研究明确治疗IgA肾病的病因和治疗方法。
microRNA（miRNA）是小的非编码RNA，在调节基因表达中起重要作用4,5。据报道，miRNA参与各种疾病的发病机制，有些已被确定为疾病生物标志物和治疗剂4,5。近年来，miRNA与IgA肾病发病机制之间的关联也有报道2,6,7。例如，miR-148b被证明与IgA肾病2,6,7患者的IgA结构异常有关，而miR-148b和let-7b被记录为检测IgA肾病的新型生物标志物7。尽管了解miRNA对IgA肾病的影响可能有助于进一步阐明病因和治疗2，但尚未建立使用小动物模型分析IgA肾病中miRNA的标准方法2。
我们在此开发了一种简单可靠的方法来测量IgA肾病小鼠模型（HIGA小鼠）肾脏中的miRNA表达水平。HIGA小鼠是一种特征性的ddY菌株，显示血清IgA水平特别高，肾小球8,9,10,11中IgA的异常沉积。因此，HIGA小鼠可用作IgA肾病小鼠模型8,9,10,11。我们的方法包括四个主要步骤：首先，通过手术从HIGA小鼠获得肾脏样本;其次，使用基于硅胶膜的离心柱均质化样品并纯化总RNA;第三，使用逆转录从总RNA合成互补DNA（cDNA）;第四，通过定量逆转录聚合酶链反应（qRT-PCR）检测miRNA的表达水平。这种方法的基本原理和结果的可靠性基于以前的报告12,13。我们表明，这是一种准确测量IgA肾病小鼠模型中miRNA表达水平的有用技术，并且可用于促进未来对IgA肾病中miRNA的研究。
Protocol
Representative Results
Discussion
我们能够使用这种新方法测量IgA肾病小鼠模型（HIGA小鼠）肾脏中miRNA的表达水平。IgA肾病是一种无法解释的疾病，需要进一步研究以阐明其病因和治疗靶点1,3。然而，获取人类肾脏样本是高度侵入性的。这种新技术的优点在于它允许使用小动物研究IgA肾病，并且应该有助于未来对IgA肾病和miRNA的研究。在未来的研究中，该方法可以应用于治疗IgA肾病的新…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢Edanz Group（www.edanzediting.com）的Sarah Williams博士编辑了这份手稿的草稿。
Materials
| BALB/cCrSlc (25-week-old, female) | Japan SLC, Inc. | none | Mouse for control |
| HIGA/NscSlc (25-week-old, female) | Japan SLC, Inc. | none | IgA nephropathy mouse model |
| MicroAmp Optical 96 well reaction plate for qRT-PCR | Thermo Fisher Scientific | 4316813 | 96-well reaction plate |
| MicroAmp Optical Adhesive Film | Thermo Fisher Scientific | 4311971 | adhesive film for 96-well reaction plate |
| miScript II RT kit | Qiagen | 218161 | Experimental kit for synthesis of cDNA |
| miRNeasy Mini kit | Qiagen | 217004 | Experimental kit fot extraction of total RNA |
| miScript Primer Assay (RNU6-2) | Qiagen | MS00033740 | miRNA-specific primer |
| miScript Primer Assay (miR-155-5p) | Qiagen | MS00001701 | miRNA-specific primer |
| miScript Primer Assay (miR-146a-5p) | Qiagen | MS00001638 | miRNA-specific primer |
| miScript Primer Assay (miR-21-5p) | Qiagen | MS00009079 | miRNA-specific primer |
| miScript SYBR Green PCR kit | Qiagen | 218073 | Experimental kit for real-time PCR |
| QIA shredder | Qiagen | 79654 | biopolymer-shredding spin column |
| QuantStudio 12K Flex Flex Real-Time PCR system | Thermo Fisher Scientific | 4472380 | real-time PCR instrument |
| QuantStudio 12K Flex Software version 1.2.1. | Thermo Fisher Scientific | 4472380 | real-time PCR instrument software |
| takara biomasher standard | Takara Bio | 9790B | silicon homogenizer |
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