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| Status |
Public on Mar 08, 2017 |
| Title |
Low-Cell-Number, Single-Tube Amplification (STA) of RNAs Revealed miRNA Changes from Pluripotency to Endothelium |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
Non-coding RNA profiling by high throughput sequencing
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| Summary |
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
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| Overall design |
total RNA from 3 types of cells was amplified and sequenced using HiSeq2500 platform
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| |
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| Contributor(s) |
Chiang P |
| Citation(s) |
28327113 |
| |
| Submission date |
Jan 16, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Po-Min Chiang |
| E-mail(s) |
[email protected]
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| Organization name |
National Cheng Kung University
|
| Department |
Institute of Clinical Medicine
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| Street address |
No. 35, Xiaodong Road,, Rm. 7068
|
| City |
Tainan |
| State/province |
TAIWAN |
| ZIP/Postal code |
70457 |
| Country |
Taiwan |
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| Platforms (1) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
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| Samples (18)
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| Relations |
| BioProject |
PRJNA361509 |
| SRA |
SRP096834 |
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