Importance of RNA isolation methods for analysis of exosomal RNA: Evaluation of different methods
Importance of RNA isolation methods for analysis of exosomal RNA: Evaluation of different methods
复制标题
DOI：
10.1016/j.molimm.2012.02.001
复制
发表时间：
2012-04-01
影响因子：
3.6
通讯作者：
Ekstrom, Karin
中科院分区：
文献类型：
--
作者：
Eldh, Maria;Lotvall, Jan;Ekstrom, Karin
外泌体是内吞起源的含有RNA的小囊泡，它可以部分通过外泌体RNA在细胞间的转移参与细胞间的通讯。外泌体由许多细胞释放，也可以在包括血浆和母乳在内的几种生物液体中发现。与其供体细胞相比，外泌体不仅在大小上不同，而且在RNA、蛋白质和脂质组成上也不同。本研究的目的是确定分析外泌体RNA的最佳RNA提取方法，以支持未来确定外泌体RNA生物学作用的研究。采用不同的方法提取外泌体和细胞RNA。采用毛细管电泳和分光光度计测定RNA完整性数（RIN）和细胞RNA OD值，评价提取的RNA的高质量和纯度。有趣的是，不同的RNA分离方法的外泌体RNA产量有很大差异。在电泳图中，外泌体RNA的模式也存在差异，表明测试方法提取的外泌体RNA具有不同的大小分布。纯柱基方法的RNA产量最高，RNA大小分布最广，而苯酚和苯酚与柱基结合的方法主要丢失较大的RNA。此外，苯酚和联合技术的使用导致外泌体RNA的产量降低，尺寸分布模式更窄，导致包括microRNA在内的小RNA富集。总之，目前的研究提出了一个独特的比较7种不同的方法提取外泌体RNA。由于不同的分离方法在外泌体RNA产量和模式上有广泛的差异，因此根据手头的研究问题选择分离方法是至关重要的。(C) 2012 Elsevier Ltd.版权所有。
Exosomes are small RNA containing vesicles of endocytic origin, which can take part in cell-to-cell communication partly by the transfer of exosomal RNA between cells. Exosomes are released by many cells and can also be found in several biological fluids including blood plasma and breast milk. Exosomes differ compared to their donor cells not only in size but also in RNA, protein and lipid composition. The aim of the current study was to determine the optimal RNA extraction method for analysis of exosomal RNA, to support future studies determining the biological roles of the exosomal RNA.Different methods were used to extract exosomal and cellular RNA. All methods evaluated extracted high quality and purity RNA as determined by RNA integrity number (RIN) and OD values for cellular RNA using capillary electrophoresis and spectrophotometer. Interestingly, the exosomal RNA yield differed substantially between the different RNA isolation methods. There was also a difference in the exosomal RNA patterns in the electropherograms, indicating that the tested methods extract exosomal RNA with different size distribution. A pure column based approach resulted in the highest RNA yield and the broadest RNA size distribution, whereas phenol and combined phenol and column based approaches lost primarily large RNAs. Moreover, the use of phenol and combined techniques resulted in reduced yield of exosomal RNA, with a more narrow size distribution pattern resulting in an enrichment of small RNA including microRNA.In conclusion, the current study presents a unique comparison of seven different methods for extraction of exosomal RNA. As the different isolation methods give extensive variation in exosomal RNA yield and patterns, it is crucial to select an isolation approach depending on the research question at hand. (C) 2012 Elsevier Ltd. All rights reserved.