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

Abstract

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.

Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification

“…The polyadenylation and ligation steps also introduce bias ( 33 – 35 ). The second group of methods includes the use of linear primers ( 36 , 37 ), pincer probes ( 38 ), and stem–loop RT primers ( 39 ), also known as TaqMan miRNA assays (Thermo Fisher). The stem–loop method is probably the most common today and is frequently used for benchmarking and validation of other methods ( 40 ).…”

qPCR-Based Methods for Expression Analysis of miRNAs

“…A universal TaqMan probe is used and the fluorescence is measured [13]. Another method was developed, which involves RT with a miRNA-specific RT primer, hybridization of the cDNA to a bi-directional extension sequence, and PCR amplification carried out with a miRNA-specific forward primer, a universal reverse primer and SYBR Green [29].…”

MicroRNA amplification and detection technologies: opportunities and challenges for point of care diagnostics

“…Northern blotting -High specificity-Readily available and easy-to-perform -Low-throughput-Low sensitivity-Laborious and very time consuming [49,86,91,92] In situ hybridization (ISH) with locked nucleic acid probes -Able to monitor the cellular and sub-cellular distributions and to determine the spatiotemporal expression profile of miRNAs -Low-throughput-Semi-quantitative analysis of miRNA expression [90] Reverse transcription qPCR (RT-qPCR) -High sensitivity and specificity -Suitable for measuring smaller miRNA panels-Can be used for absolute quantification-Easy to incorporate into the workflow for laboratories that are used to qPCR-Suitable for routine measurements in laboratory settings-Can be automated to a high degree -Cannot identify novel miRNAs -Medium-throughput with respect to the number of samples that can be processed per day -Optimal reaction conditions may differ considerably between miRNAs due to sequence-specific differences in primer annealing [29,32,36,86,87] Microarray -Rather low-cost and high-throughput considering the number of samples that can be processed per day -Suitable for comparing the relative abundance of specific miRNAs between two states (e.g., 'diseased' versus 'non-diseased) -Low sensitivity and specificity -Long turnaround time-Poor degree of automation-Not suitable for absolute quantification-Cannot identify novel miRNAs [41,[86][87][88] Next generation sequencing (NGS) -Widest range of applications -Very high sensitivity-High accuracy in distinguishing variants of miRNAs that are very similar in sequence -Substantial computational support needed for data analysis-Cannot be used for absolute quantification-Rather expensive and not available everywhere-Not so suitable for high-throughput laboratory settings yet [50,86,87,89] [28]. NB analysis has the advantage of simultaneously detecting the mature miRNAs and miRNA precursors, but it is expensive, very labor-intensive and time-consuming approach which also requires radio-labeling.…”