Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering , Shaanxi Normal University , Xi'an 710119 , Shaanxi Province , P. R. China.
ACS Sens. 2018 Sep 28;3(9):1795-1801. doi: 10.1021/acssensors.8b00524. Epub 2018 Sep 5.
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.
精确检测单个细胞中信使 RNA（mRNA）突变的低拷贝数具有重要意义，但仍然具有挑战性。在此，通过整合精心设计的肽核酸（PNA）夹的优异特性，该夹可高度选择性地区分单核苷酸变异，以及用于超灵敏和精确定量的液滴数字 PCR，我们开发了一种稳健的一步液滴数字逆转录 PCR（ddRT-PCR）方法，该方法可在超高背景下的野生型 mRNA 中，以超高特异性精确检测单个细胞中的 mRNA 突变，可清晰分辨低至 0.01%的突变 mRNA。由于其出色的单分子水平灵敏度和超高特异性，这种 ddRT-PCR 方法有望用于在单细胞水平上研究细胞异质性，以及用于在复杂的血浆或血清中对突变 mRNA 进行精确定量，从而实现液体活检。
