Retrovirus Laboratory, Department of Molecular and Comparative Pathobiology, The Johns Hopkins University School of Medicine Baltimore, MD, USA.
Front Genet. 2013 May 16;4:83. doi: 10.3389/fgene.2013.00083. eCollection 2013.
Interest in extracellular RNA (exRNA) has intensified as evidence accumulates that these molecules may be useful as indicators of a wide variety of biological conditions. To establish specific exRNA molecules as clinically relevant biomarkers, reproducible recovery from biological samples and reliable measurements of the isolated RNA are paramount. Toward these ends, careful and rigorous comparisons of technical procedures are needed at all steps from sample handling to RNA isolation to RNA measurement protocols. In the investigations described in this methods paper, RT-qPCR was used to examine the apparent recovery of specific endogenous miRNAs and a spiked-in synthetic RNA from blood plasma samples. RNA was isolated using several widely used RNA isolation kits, with or without the addition of glycogen as a carrier. Kits examined included total RNA isolation systems that have been commercially available for several years and commonly adapted for extraction of biofluid RNA, as well as more recently introduced biofluids-specific RNA methods. Our conclusions include the following: some RNA isolation methods appear to be superior to others for the recovery of RNA from biological fluids; addition of a carrier molecule seems to be beneficial for some but not all isolation methods; and quantitative recovery of RNA is observed from increasing volumes of cerebrospinal fluid.
人们对细胞外 RNA（exRNA）的兴趣日益浓厚，因为越来越多的证据表明，这些分子可能作为各种生物状况的指标很有用。为了将特定的 exRNA 分子确立为具有临床相关性的生物标志物，从生物样本中进行可重复的回收以及对分离的 RNA 进行可靠的测量至关重要。为此，需要在从样品处理到 RNA 分离再到 RNA 测量方案的所有步骤中，对技术程序进行仔细和严格的比较。在本方法论文中描述的研究中，使用 RT-qPCR 检查了特定内源性 miRNA 和从血浆样品中掺入的合成 RNA 的明显回收率。使用几种广泛使用的 RNA 分离试剂盒从血液或其他体液中分离 RNA，这些试剂盒有的添加糖原作为载体，有的则不添加。检查的试剂盒包括已经商业化多年并且通常适用于提取生物流体 RNA 的总 RNA 分离系统，以及最近推出的特定于生物流体的 RNA 方法。我们的结论包括以下内容：某些 RNA 分离方法在从生物流体中回收 RNA 方面似乎优于其他方法；添加载体分子似乎对某些但不是所有的分离方法都有益；并且从增加的脑脊液体积中观察到 RNA 的定量回收。
