Persson Helena, Søkilde Rolf, Pirona Anna Chiara, Rovira Carlos
Lund University Cancer Center, Faculty of Medicine, Department of Clinical Sciences Lund, Oncology and Pathology, Lund, Sweden.
BioCARE, Strategic Cancer Research Program, Lund, Sweden.
Biotechniques. 2017 Aug 1;63(2):57-64. doi: 10.2144/000114574.
MicroRNAs (miRNAs) are ~22-nucleotide-long small non-coding RNAs that regulate the expression of protein-coding genes by base pairing to partially complementary target sites, preferentially located in the 3´ untranslated region (UTR) of target mRNAs. The expression and function of miRNAs have been extensively studied in human disease, as well as the possibility of using these molecules as biomarkers for prognostication and treatment guidance. To identify and validate miRNAs as biomarkers, their expression must be screened in large collections of patient samples. Here, we develop a scalable protocol for the rapid and economical preparation of a large number of small RNA sequencing libraries using dual indexing for multiplexing. Combined with the use of off-the-shelf reagents, more samples can be sequenced simultaneously on large-scale sequencing platforms at a considerably lower cost per sample. Sample preparation is simplified by pooling libraries prior to gel purification, which allows for the selection of a narrow size range while minimizing sample variation. A comparison with publicly available data from benchmarking of miRNA analysis platforms showed that this method captures absolute and differential expression as effectively as commercially available alternatives.
微小RNA（miRNA）是长度约为22个核苷酸的小型非编码RNA，通过与部分互补的靶位点碱基配对来调节蛋白质编码基因的表达，这些靶位点优先位于靶mRNA的3´非翻译区（UTR）。miRNA的表达和功能已在人类疾病中得到广泛研究，以及将这些分子用作预后和治疗指导生物标志物的可能性。为了鉴定和验证miRNA作为生物标志物，必须在大量患者样本中筛选它们的表达。在这里，我们开发了一种可扩展的方案，用于使用双索引进行多路复用，快速且经济地制备大量小RNA测序文库。结合使用现成的试剂，可以在大规模测序平台上同时对更多样本进行测序，每个样本的成本大大降低。通过在凝胶纯化之前合并文库来简化样本制备，这允许选择较窄的大小范围，同时最小化样本变异。与来自miRNA分析平台基准测试的公开可用数据进行比较表明，该方法与市售替代方法一样有效地捕获绝对表达和差异表达。
