使用定量实时PCR检测大鼠腹膜中的微小RNA表达
Summary
在这里，我们提出了使用定量实时逆转录聚合酶链反应检测大鼠腹膜中microRNA表达的方案。该方法适用于在几种病理条件下研究大鼠腹膜中的microRNA表达谱。
Abstract
微小RNA（miRNA）是在转录后调节信使RNA表达的小型非编码RNA。已经在大鼠的各种器官和组织中研究了miRNA表达谱。然而，用于纯化miRNA的标准方法和其在大鼠腹膜中的表达检测尚未很好地建立。我们已经开发了一种有效和可靠的方法，使用定量实时逆转录聚合酶链反应（qRT-PCR）在大鼠腹膜中纯化和定量miRNA。该方案由四个步骤组成：1）净化腹膜样品; 2）从腹膜样品纯化包括miRNA的总RNA; 3）逆转录miRNA产生cDNA;和4）qRT-PCR检测miRNA表达。使用该方案，我们成功地确定了6种miRNA（miRNA-142-3p，miRNA-21-5p，miRNA-221-3p，miRNA-223-3p，miRNA-327和miRNA-34a-5p）的表达增加与对照组相比，大鼠腹膜纤维化模型的腹膜显着。该方案可用于研究许多病理条件下大鼠腹膜中miRNA表达谱。
Introduction
微小RNA（miRNA）是转录后调节信使RNA（mRNA）表达的短的非编码RNA。 miRNAs表达的变化调节许多在各种病理状况（包括癌症，炎症，代谢紊乱和纤维化）2,3,4,5,6,7,8中发挥关键作用的mRNA的表达。因此，miRNA具有潜在的新生物标志物和治疗靶标2,3,4,5,6,7,8。已经在各种大鼠中测定了miRNA表达谱器官和组织，包括肝脏，心脏，肺和肾脏9 。然而，用于纯化和检测大鼠腹膜中的miRNA的标准方法尚未得到很好的建立。
该方案的总体目标是成功地纯化和检测大鼠腹膜中的miRNA。首先，使用玻璃均化器将腹膜样品匀浆，然后在微量离心旋转塔10中暴露于生物聚合物破碎系统。接下来，使用基于二氧化硅膜的旋转柱10从腹膜样品纯化包括miRNA的总RNA。然后，使用逆转录酶，poly（A）聚合酶和oligo-dT引物11从纯化的总RNA合成cDNA。最后，使用插层染料11通过qRT-PCR测定miRNA的表达。这个协议的理由是b在以前的研究中，通过简单的过程8,10,11，显示了组织中miRNA的显着纯化和检测。据报道，在微量离心旋转柱和基于二氧化硅膜的旋转柱中使用生物聚合物粉碎系统可以从组织10中纯化高质量的总RNA。已经报道了使用逆转录酶，poly（A）聚合酶和oligo-dT引物从纯化的总RNA合成cDNA的方法，以及通过使用插入染料通过qRT-PCR检测miRNA表达的方法已经报道显示出高精度和灵敏度11 。此外，这是一个简单的过程，节省时间并防止技术错误。因此，该方案在需要在大范围病理条件下对大鼠腹膜中的miRNA进行高度准确和灵敏的检测的研究中是有用的。
Protocol
Representative Results
Discussion
使用本手册中提出的方案，大鼠腹膜中的miRNA成功纯化并使用qRT-PCR进行检测。 qRT-PCR数据分析的可靠性取决于纯化的miRNA的质量。因此，可以在qRT-PCR前通过260nm处的吸光度与280nm处的吸光度比检查miRNA的纯度，这可以使用分光光度计测量。当使用qRT-PCR不能获得miRNA的显着扩增时，模板cDNA的浓度可能会增加。每个miRNA特异性引物的浓度也可以增加。
确定miRNA表达水平有几种替代方?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们要感谢Miyako Shigeta的出色的技术支持。这项工作得到了JSS KAKENHI（授权号25461252）的部分支持。
Materials
| QIA shredder | Qiagen | 79654 | biopolymer-shredding system in a micro centrifuge spin-column |
| miRNeasy Mini kit | Qiagen | 217004 | silica-membrane based spin column |
| QIAzol Lysis Reagent | Qiagen | 79306 | phenol/guanidine-based lysis reagent |
| Buffer RLT | Qiagen | 79216 | wash buffer 1 |
| Buffer RWT | Qiagen | 1067933 | wash buffer 2 |
| miScript II RT kit | Qiagen | 218161 | includes 10× Nucleics Mix containing deoxynucleotides, ribonucleotide triphosphates, and oligo-dT primers; miScript Reverse Transcriptase Mix containing poly(A) polymerase and reverse transcriptase and; miScript HiSpec buffer |
| miScript SYBR Green PCR kit | Qiagen | 218073 | includes QuantiTect SYBR Green PCR Master Mix and miScript Universal Primer |
| RNU6-2 primer | Qiagen | MS00033740 | not disclosed |
| miRNA-142-3p primer | Qiagen | MS00031451 | 5'-UGUAGUGUUUCC UACUUUAUGGA-3' |
| miRNA-21-5p primer | Qiagen | MS00009079 | 5'-UAGCUUAUCAG ACUGAUGUUGA-3' |
| miRNA-221-3p primer | Qiagen | MS00003857 | 5'-AGCUACAUUGU CUGCUGGGUUUC-3' |
| miRNA-223-3p primer | Qiagen | MS00033320 | 5'-UGUCAGUUUG UCAAAUACCCC-3' |
| miRNA-34a-5p primer | Qiagen | MS00003318 | 5'-UGGCAGUGUCU UAGCUGGUUGU-3' |
| miRNA-327 primer | Qiagen | MS00000805 | 5'-CCUUGAGGGG CAUGAGGGU-3' |
| 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 |
| 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 |
| methylglyoxal | Sigma-Aldrich | M0252 | |
| Midperic | Terumo | not assign | peritoneal dialysis fluid |
| Sprague–Dawley rats | SLC | not assign |
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