Quantification of Mature MicroRNAs Using Pincer Probes and Real-Time PCR Amplification

The robust and reliable detection of small microRNAs (miRNAs) is important to understand the functional significance of miRNAs. Several methods can be used to quantify miRNAs. Selectively quantifying mature miRNAs among miRNA precursors, pri-miRNAs, and other miRNA-like sequences is challenging because of the short length of miRNAs. In this study, we developed a two-step miRNA quantification system based on pincer probe capture and real-time PCR amplification. The performance of the method was tested using synthetic mature miRNAs and clinical RNA samples. Results showed that the method demonstrated dynamic range of seven orders of magnitude and sensitivity of detection of hundreds of copies of miRNA molecules. The use of pincer probes allowed excellent discrimination of mature miRNAs from their precursors with five Cq (quantification cycle) values difference. The developed method also showed good discrimination of highly homologous family members with cross reaction less than 5%. The pincer probe-based approach is a potential alternative to currently used methods for mature miRNA quantification.