Micro RNAs(mi RNAs) have attracted significant attention in biomedical research and clinical diagnosis.However, due to their inherent characteristics of low abundance and the high complexity of corresponding biologica...Micro RNAs(mi RNAs) have attracted significant attention in biomedical research and clinical diagnosis.However, due to their inherent characteristics of low abundance and the high complexity of corresponding biological matrices, simultaneous detection of multiple mi RNAs at low abundance is still a challenge.In this work, a method coupling exponential amplification reaction(EXPAR) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS) is developed for label-free and simultaneous detection of multiple mi RNAs. The assay can be performed under isothermal conditions in a single reaction tube, and finished in less than 30 min. It exhibits good quantification ability and with attomolar-level sensitivity for mi RNAs detection. It also shows high specificity to distinguish mi RNAs at single-nucleotide resolution. We used the method to detect the mi RNA-21, let-7a, mi RNA-100, and mi RNA-125b in samples of spiked human serum and breast cancer cells(i.e., MCF-7, MDA-MB-231 and SK-BR-3). The quantification results were well consistent with the standard real-time fluorescence EXPAR.Consequently, the label-free mass-spectrometric platform could be a potential tool for mi RNAs analysis in complex biological samples, and may be used for clinical diagnosis.展开更多
基金supported by the National Natural Science Foundation of China (NSFC, Nos. 22022401, 22074022 and 21934001)the Ministry of Science and Technology of China (Nos. 2020YFF0426500, 2020YFF0304502)。
文摘Micro RNAs(mi RNAs) have attracted significant attention in biomedical research and clinical diagnosis.However, due to their inherent characteristics of low abundance and the high complexity of corresponding biological matrices, simultaneous detection of multiple mi RNAs at low abundance is still a challenge.In this work, a method coupling exponential amplification reaction(EXPAR) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS) is developed for label-free and simultaneous detection of multiple mi RNAs. The assay can be performed under isothermal conditions in a single reaction tube, and finished in less than 30 min. It exhibits good quantification ability and with attomolar-level sensitivity for mi RNAs detection. It also shows high specificity to distinguish mi RNAs at single-nucleotide resolution. We used the method to detect the mi RNA-21, let-7a, mi RNA-100, and mi RNA-125b in samples of spiked human serum and breast cancer cells(i.e., MCF-7, MDA-MB-231 and SK-BR-3). The quantification results were well consistent with the standard real-time fluorescence EXPAR.Consequently, the label-free mass-spectrometric platform could be a potential tool for mi RNAs analysis in complex biological samples, and may be used for clinical diagnosis.
