Yan Yan, Chang Chi-Chih, Venø Morten T, Mothershead Colin R, Su Junyi, Kjems Jørgen
The Interdisciplinary Nanoscience Centre, Aarhus University.
The Interdisciplinary Nanoscience Centre, Aarhus University; Department of Molecular Biology and Genetics, Aarhus University.
J Vis Exp. 2019 Mar 8(145). doi: 10.3791/58655.
Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators - microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.
细胞外和循环RNA（exRNA）由身体的多种细胞类型产生，存在于多种体液中，如唾液、血浆、血清、乳汁和尿液。这些RNA的一个子集是转录后调节因子——微小RNA（miRNA）。为了描绘特定细胞类型产生的miRNA，可使用体外培养系统来收集和分析源自一类细胞的exRNA。间充质干细胞的分泌因子与多种疾病的缓解有关，本文将其用作体外模型系统。本文描述了收集、纯化小RNA以及构建文库以对细胞外miRNA进行测序的过程。培养基中的exRNA与细胞RNA不同，其RNA输入量低，这就需要优化流程。本方案为从培养基中进行小exRNA测序提供了全面指南，展示了exRNA纯化和测序过程中每个步骤的质量控制检查点。
