Identification of extracellular miRNA in human cerebrospinal fluid by next-generation sequencing
Identification of extracellular miRNA in human cerebrospinal fluid by next-generation sequencing
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DOI：
10.1261/rna.036863.112
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发表时间：
2013-05-01
期刊：
影响因子：
4.5
通讯作者：
Van Keuren-Jensen, Kendall
中科院分区：
文献类型：
--
作者：
Burgos, Kasandra Lovette;Javaherian, Ashkan;Van Keuren-Jensen, Kendall
人们越来越有兴趣使用新一代测序 (NGS) 来分析神经系统疾病、中枢神经系统肿瘤或创伤性脑损伤患者的血液和脑脊液 (CSF) 中的细胞外小 RNA，以发现生物标志物。小样本量和低 RNA 丰度的样本给下游小 RNA 测序分析带来了挑战。血浆、血清和脑脊液含有少量的总 RNA，其中小 RNA 占一小部分。本研究的目的是最大限度地从 RNA 有限的样本中分离出 RNA，并应用这些方法通过小 RNA 深度测序来分析人类 CSF 中的 miRNA。我们使用十种市售试剂盒系统地测试了 RNA 分离效率，并比较了它们在人血浆样本上的性能。我们使用 RiboGreen 量化总 RNA 产量，并使用定制 TaqMan 测定来确定每个试剂盒的小 RNA 分离效率。我们通过在性能最佳的试剂盒中进行苯酚-氯仿纯化过程中重复水提取，显着提高了小 RNA 的回收率。随后，我们使用小 RNA 产量最高的方法从同一个体的脑脊液和血清样本中纯化 RNA。然后，我们使用 Illumina 的 TruSeq 样品制备试剂盒制备小 RNA 测序文库，并在 HiSeq 2000 上对样品进行测序。毫不奇怪，我们发现 CSF 的 miRNA 表达谱与血清的 miRNA 表达谱有很大不同。据我们所知，这是第一次使用新一代测序技术对脑脊液中的小 RNA 片段进行分析。
There has been a growing interest in using next-generation sequencing (NGS) to profile extracellular small RNAs from the blood and cerebrospinal fluid (CSF) of patients with neurological diseases, CNS tumors, or traumatic brain injury for biomarker discovery. Small sample volumes and samples with low RNA abundance create challenges for downstream small RNA sequencing assays. Plasma, serum, and CSF contain low amounts of total RNA, of which small RNAs make up a fraction. The purpose of this study was to maximize RNA isolation from RNA-limited samples and apply these methods to profile the miRNA in human CSF by small RNA deep sequencing. We systematically tested RNA isolation efficiency using ten commercially available kits and compared their performance on human plasma samples. We used RiboGreen to quantify total RNA yield and custom TaqMan assays to determine the efficiency of small RNA isolation for each of the kits. We significantly increased the recovery of small RNA by repeating the aqueous extraction during the phenol-chloroform purification in the top performing kits. We subsequently used the methods with the highest small RNA yield to purify RNA from CSF and serum samples from the same individual. We then prepared small RNA sequencing libraries using Illumina's TruSeq sample preparation kit and sequenced the samples on the HiSeq 2000. Not surprisingly, we found that the miRNA expression profile of CSF is substantially different from that of serum. To our knowledge, this is the first time that the small RNA fraction from CSF has been profiled using next-generation sequencing.