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| Status |
Public on Oct 03, 2017 |
| Title |
A radiolabeling-free, qPCR-based method for locus-specific pseudouridine detection |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
Pseudouridine (Ψ) is the most abundant post-transcriptional RNA modification. Various methods have been developed to achieve locus-specific Ψ detection; however, the existing methods often involve radiolabeling of RNA, require advanced experimental skills and can be time-consuming. Herein we report a radiolabeling-free，qPCR-based method to detect locus-specific Ψs in rRNA and mRNA. This method is based on Ψ chemical adduct (Ψ-CMC) induced mutation/deletion during reverse transcription (RT), leading to qPCR products of different melting temperatures. Utilizing high-throughput sequencing, we demonstrate that such misincorporation is a general feature of Ψ-CMC adduct during our improved RT conditions. We validated this method on known Ψ sites in rRNA and showed that the melting curves correlate with the modification level. Moreover, we successfully detected Ψs in mRNA and lncRNA of different abundance, and identified Ψ synthase that targets mRNA. Our facile method takes only 1.5 days to complete, and with slight adjustment it can be applied to detect other epitranscriptomic marks in the transcriptome.
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| Overall design |
Here we report a radiolabeling-free, qPCR based method to rapidly detect Ψ in a locus-specific manner. The method is based on Ψ labelling adduct induced misincorporation during reverse transcription (RT), generating qPCR products of different melting temperatures. To demonstrate that read-through induced misincorporation is a general feature of Ψ sites under our improved RT condition, we utilized high-throughput sequencing to comprehensively examine the pattern of misincorporation for all Ψ sites in rRNA using HEK293T cells. Besides, to address the detection resolution of the method, three qPCR amplicons examined with our locus-specific approach were subjected to high-throughput sequencing (for MALAT1, EEF1A1 and 18S rRNA).
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| Contributor(s) |
Lei Z, Yi C |
| Citation(s) |
28960747 |
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| Submission date |
Aug 10, 2017 |
| Last update date |
Jul 25, 2021 |
| Contact name |
Chengqi Yi |
| E-mail(s) |
[email protected]
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| Organization name |
Peking University
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| Street address |
5 Yiheyuan Road, Haidian District
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| City |
Beijing |
| ZIP/Postal code |
100871 |
| Country |
China |
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| Platforms (1) |
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| Samples (12)
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| Relations |
| BioProject |
PRJNA397844 |
| SRA |
SRP115188 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE102476_RAW.tar |
1.1 Mb |
(http)(custom) |
TAR (of TXT) |
| GSE102476_Stat_CMC+_deletion.txt.gz |
98.9 Kb |
(ftp)(http) |
TXT |
| GSE102476_Stat_stop_rates_difference.txt.gz |
118.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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