Lee Yi-Hsuan, Hsueh Ya-Wen, Peng Yao-Hung, Chang Kung-Chao, Tsai Kuen-Jer, Sun H Sunny, Su Ih-Jen, Chiang Po-Min
Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan.
Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, No. 35, Xiaodong Rd, Tainan, 70457, Taiwan.
BMC Biol. 2017 Mar 21;15(1):22. doi: 10.1186/s12915-017-0359-5.
In addition to messenger RNA (mRNA), noncoding RNAs (ncRNAs) are essential components in cellular machineries for translation and splicing. Besides their housekeeping functions, ncRNAs are involved in cell type-specific regulation of translation, mRNA stability, genome structure, and accessibility. To have a comprehensive understanding of the identities and functions of different cell types, a method to comprehensively quantify both mRNA and ncRNA in a sensitive manner is highly desirable.
Here we tried to develop a system capable of concurrently profiling both mRNA and ncRNA by polyadenylating RNA in samples before reverse transcription. The sensitivity of the system was maximized by avoiding purification from cell lysis to amplified cDNA and by optimizing the buffer conditions. The single-tube amplification (STA) system was applied to single to 100 cells of 293T cells, human pluripotent stem cells (hPSCs) and their differentiated endothelial progenies to validate its quantitative power and sensitivity by qPCR and high-throughput sequencing.
Using microRNA (miRNA) as an example, we showed that complementary DNA (cDNA) from ncRNAs 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 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. Finally, the STA system was capable of detecting miRNA and mRNA expression down to single cells, albeit with some loss of sensitivity and power.
Overall, STA offered a simple and sensitive way to concurrently quantify both mRNA and ncRNA expression in low-cell-number samples for both qPCR and high-throughput sequencing.
除信使核糖核酸（mRNA）外，非编码核糖核酸（ncRNAs）是细胞翻译和剪接机制的重要组成部分。除了其管家功能外，ncRNAs还参与细胞类型特异性的翻译调控、mRNA稳定性、基因组结构和可及性。为了全面了解不同细胞类型的特性和功能，非常需要一种能够以灵敏方式同时定量mRNA和ncRNA的方法。
在此，我们试图开发一种系统，该系统能够通过在逆转录前对样品中的RNA进行聚腺苷酸化来同时分析mRNA和ncRNA。通过避免从细胞裂解到扩增cDNA的纯化过程并优化缓冲液条件，使该系统的灵敏度最大化。将单管扩增（STA）系统应用于293T细胞、人多能干细胞（hPSCs）及其分化的内皮后代的单个至100个细胞，通过定量聚合酶链反应（qPCR）和高通量测序来验证其定量能力和灵敏度。
以微小核糖核酸（miRNA）为例，我们表明ncRNAs的互补DNA（cDNA）可以在单管内从少数细胞中扩增并特异性检测到。通过避免从细胞裂解到扩增cDNA的纯化过程并优化缓冲液条件，使该系统的灵敏度最大化。以100个人类胚胎干细胞（hESCs）及其分化的内皮细胞作为高通量测序的输入，单管扩增（STA）系统揭示了在每种细胞类型中选择性富集的知名miRNA和其他miRNA。通过对293FT细胞和一个人诱导多能干细胞（hiPSC）系进行qPCR，进一步验证了miRNA的选择性富集。此外，对其他非miRNA转录本的检测表明，STA的靶标不仅限于miRNA，还扩展到了其他ncRNAs和mRNAs。最后，STA系统能够检测低至单个细胞的miRNA和mRNA表达，尽管灵敏度和效能有所损失。
总体而言，STA为通过qPCR和高通量测序同时定量低细胞数样品中的mRNA和ncRNA表达提供了一种简单而灵敏的方法。
