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Analysis of microRNAs by the Stem-Loop Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction Using A Double Quencher Probe


 
 
 We created a universal probe that can be used with mature micro RNAs (miRNAs) using a double quencher incorporating the ZEN quencher into the hydrolysis probe required for miRNA analysis by the real- time quantitative polymerase chain reaction (RT-qPCR) using a stem-loop primer. The main purpose of this approach was to improve sensitivity and convenience.
 
 
 
 We conducted studies on miR-21, miR-30a and miR-200c. To assess the relationship between the sequence interval and reactivity between the miRNA-specific sequence on the stem-loop primer and detection probe, various sequence patterns were examined using miRNA from K562 cells. The sensitivity and reactivity of the designed primers and probes were examined using each synthetic miRNA and transurethral resection of bladder tumor (TURBT) samples. The synthetic RNA was serially diluted in 10% increments. Twenty-seven TURBT cases were examined. The prepared samples were extracted with the FastGene RNA Basic Kit and FastGene miRNA Enhancer. After extraction, cDNA was prepared using FastGene Scriptase II. We performed quantitative analysis by the RT-qPCR using a ZEN double quencher probe. The samples were analyzed with the Taq Man miRNA assay.
 
 
 
 The probe and miRNA-specific sequences of the stem-loop primer showed better reactivity when the design contained less space within the sequences. Serial dilution of the samples using synthetic miRNA showed that linearity depended on the addition amount, and occurred at a concentration of log 7 or more. In the TURBT samples, the Δ of the mean Cp value, and the correlation coefficients of miR-21, miR-30a, and miR-200c, were 3.13 and 0.966; 1.10 and 0.991; and 0.39 and 0.997, respectively, compared with the TaqMan miRNA assay.
 
 
 
 This demonstrates that our method has the same or higher sensitivity than the TaqMan miRNA assay. Overall, these findings suggest that the analysis of miRNA with a stem-loop primer using the double quencher probe is useful.
