Sensitive profiling of noncoding transcriptome from pluripotency to hemogenic endothelium

• Main Authors: Yu-TungChiu, 邱語彤
• Other Authors: Po-Min Chiang
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/7ek3u4

碩士 === 國立成功大學 === 臨床醫學研究所 === 105 === SUMMARY In addition to messenger RNA (mRNA), non-coding RNA (ncRNA) plays an important role in cells. miRNAs’ function is to regulate different function of cells, stability of mRNA and genome structure. There are many ways to detect or analyze miRNAs, such as quantitative reverse transcription polymerase chain reaction (qRT-PCR), microarray, and next-generation sequencing (NGS). However, these methods have low sensitivity and require large numbers of cells to detect miRNA. So we wanted to find a more sensitive detection system. We used polyadenylation to analyze mRNA and ncRNA in cells, simultaneously. Then we adjusted the ion concentration in buffer to increase the sensitivity of the detection system. We applied this system to low number or single HEK293 cells and human pluripotency stem cells (hPSCs), and then confirmed the sensitivity of the system with qRT-PCR and high throughput sequencing. According to the results, we can observe the different type of RNA in low number or single cells. Finally, the selective enrichment of HEK293 cells and hPSC cells was verified by qRT-PCR. We found that the system can detect miRNA in addition, but also can analyze other ncRNA and mRNA. Overall, we have established a simple and sensitive way to concurrently quantify both mRNA and ncRNA in low cell number for qRT-PCR and high throughput sequencing.