Journal
ACS SENSORS
Volume 3, Issue 9, Pages 1795-1801Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssensors.8b00524
Keywords
mRNA mutation; PNA clamp; ddPCR; single cell analysis; analytical methods
Funding
- National Natural Science Foundation of China [21335005, 21622507, 21472120]
- Program for Changjiang Scholars and Innovative Research Team in University [IRT 15_R43]
- Fundamental Research Funds for the Central Universities [GK201802016]
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Precise detection of the low copy numbers of messenger RNA (mRNA) mutation in single cells is of great significance but still remains challenging. Herein, by integrating the outstanding features of a rationally designed peptide nucleic acid (PNA) clamp for highly selective discrimination of single-nucleotide variation, and droplet digital PCR for ultrasensitive and precise quantification, we have developed a robust one-step droplet digital reverse transcription PCR (ddRT-PCR) method which enables precise mRNA mutation detection in single cells with ultrahigh specificity to clearly discern as low as 0.01% mutated mRNA in a high background of wild-type mRNA. Because of its outstanding single-molecule level sensitivity and ultrahigh specificity, this ddRT-PCR method holds great promise for studying cellular heterogeneity at the single cell level, as well as for the precise quantification of mutant mRNAs in complex plasma or serum for liquid biopsy.
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