Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA.
Analyst. 2017 Aug 21;142(17):3203-3211. doi: 10.1039/c7an00670e.
MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression at the post-transcriptional level via a complex regulatory network that requires genome-wide miRNA profiling to dissect. The patterns of miRNA expression at the genome scale are rich in diagnostic and prognostic information for human diseases such as cancers. This analysis, however, requires multi-step purification of RNAs from large quantities of cells, which is not only time consuming and costly but also challenging in situations where cell numbers are limited. In this study, we report direct capture, amplification, and library preparation of miRNAs from whole cell lysate without the need of pre-purification. As a result, it enables genome-wide miRNA profiling reproducibly with low quantity of cell samples (∼500 hematopoietic cells). Specifically, we conducted a systematic investigation of two key steps - cell lysis for miRNA release and 3' adaptor ligation required for direct miRNA capture and amplification. The obtained expression profile not only distinguishes cell types but also detects individual miRNA alterations in closely related isogenic cell lines. This approach, which is substantially simple as compared to the standard methods because of elimination of the need for RNA purification, is advantageous for the measurement of low quantity samples.
微小 RNA（miRNAs）是一类小型非编码 RNA，通过复杂的调控网络在转录后水平上控制基因表达，需要进行全基因组 miRNA 图谱分析来解析。miRNA 在基因组范围内的表达模式富含人类疾病（如癌症）的诊断和预后信息。然而，这种分析需要从大量细胞中多次纯化 RNA，不仅耗时且昂贵，而且在细胞数量有限的情况下也具有挑战性。在这项研究中，我们报告了无需预纯化即可直接从全细胞裂解物中捕获、扩增和制备 miRNA 的方法。其结果是，可以使用低数量的细胞样本（约 500 个造血细胞）可靠地进行全基因组 miRNA 图谱分析。具体而言，我们对两个关键步骤进行了系统研究——释放 miRNA 所需的细胞裂解，以及直接 miRNA 捕获和扩增所需的 3'衔接子连接。所得表达谱不仅可以区分细胞类型，还可以检测到密切相关的同基因细胞系中单个 miRNA 的变化。与标准方法相比，由于不需要 RNA 纯化，这种方法大大简化，对于低数量样本的测量具有优势。
