Using PNA Probes for Hybridization-Based Analysis of miRNAs in Capillary Electrophoresis
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The inherent separation inefficiencies of the excess probes and hybrids and the hybrids from each other limits the use of capillary electrophoresis (CE) for hybridization-based analysis of microRNA (miRNAs). Our lab developed direct quantitative analysis of multiple miRNA (DQAMmiR) to address the above using two types of mobility shifters: single-strand DNA binding protein (SSB) added to the CE run buffer and peptide drag tags conjugated with the probes. Here, we introduce second-generation DQAMmiR, which is simpler than the first-generation as it omits the addition of SSB protein in CE run buffer by the use of uncharged Peptide Nucleic Acid (PNA) instead of DNA probes. Additionally, the performance of the assay was also validated in the presence of prostate cancer (PCa)-derived crude cell lysate for two deregulated miRNAs. With ongoing improvements, PNA-facilitated DQAMmiR holds great potential for reliable direct quantitative analysis of endogenous miRNAs in clinical samples.