Low-Cell-Number, Single-Tube Amplification (STA) of RNAs Revealed miRNA Changes from Pluripotency to Endothelium. Homo sapiens

Non-coding RNAs (ncRNAs) are essential components in cellular machineries for translation and splicing. In addition to their housekeeping functions, ncRNAs are involved in cell type-specific regulation of translation, mRNA stability, genome structure and accessibility. To decipher their functions in different cell types, a method to comprehensively quantify them in a sensitive manner is highly desirable. Using miRNA as an example, we showed that cDNA from total RNA could be amplified and specifically detected from a few cells within a single tube. The sensitivity of the system was maximized by avoiding purification from cell lysis to amplified cDNA and by optimizing the buffer conditions. With 100 human embryonic stem cells (hESCs) and their differentiated endothelial cells as input for high-throughput sequencing, the single-tube amplification (STA) system revealed both well-known and other miRNAs selectively enriched in each cell type. The selective enrichment of the miRNAs was further verified by qPCR with 293FT cells and a human induced pluripotent stem cell (hiPSC) line. In addition, the STA system was capable of detecting miRNA expression down to single cells, albeit with some loss of sensitivity and power. Finally, the detection of other non-miRNA transcripts indicated that the STA target was not limited to miRNA, but extended to other ncRNAs and mRNAs as well. Overall, STA offered a simple and extremely sensitive way to collect the quantitative miRNA and other RNA information from individual cells. Grant ID: MOST-104-2314-B-006-038-MY3 Funding source: Ministry of Science and Technology, Taiwan Grantee First Name: Po-Min Grantee Last Name: Chiang Overall design: total RNA from 3 types of cells was amplified and sequenced using HiSeq2500 platform

非编码RNA（non-coding RNAs，ncRNAs）是细胞内参与翻译与剪接过程的关键组分。除持家功能外，非编码RNA还参与细胞类型特异性的翻译调控、mRNA稳定性调控、基因组结构与可及性调控。为解析其在不同细胞类型中的功能，亟需建立一种可灵敏且全面定量此类RNA的方法。以微小RNA（microRNA，miRNA）为例，本研究证实，总RNA逆转录得到的cDNA可在单管体系中从少量细胞内完成扩增并实现特异性检测。通过规避从细胞裂解至扩增cDNA阶段的纯化步骤并优化缓冲液条件，该体系的灵敏度得以最大化。以100个人类胚胎干细胞（human embryonic stem cells，hESCs）及其分化的内皮细胞作为高通量测序的起始样本，单管扩增（single-tube amplification，STA）体系揭示了两类细胞中既存的已知miRNA，以及其他细胞类型特异性富集的miRNA。该miRNA的选择性富集现象进一步通过293FT细胞及一株人类诱导多能干细胞（human induced pluripotent stem cell，hiPSC）的qPCR实验得到验证。此外，尽管灵敏度与检测效能略有损失，STA体系仍可实现单个细胞水平的miRNA表达检测。针对其他非miRNA转录本的检测结果进一步表明，STA的靶标并不局限于miRNA，而是可拓展至其他非编码RNA及mRNA。总体而言，STA提供了一种简便且极高灵敏度的方法，可从单个细胞中获取定量的miRNA及其他RNA信息。资助编号：MOST-104-2314-B-006-038-MY3
资助方：中国台湾科技部
受资助人：Po-Min Chiang
整体实验设计：对三种细胞类型的总RNA进行扩增，并采用HiSeq2500平台完成测序。