title: Improved RT-PCR Assay to Quantitate the Pri-, Pre-, and Mature microRNAs with Higher Efficiency and Accuracy.
abstract: Understanding of the functional significance of microRNAs (miRNAs) requires efficient and accurate detection method. In this study, we developed an improved miRNAs quantification system based on quantitative real-time polymerase chain reaction (qRT-PCR). This method showed higher efficiency and accuracy to survey the expression of primary miRNAs (pri-miRNAs), precursor miRNAs (pre-miRNAs), and mature miRNAs. Instead of relative quantification method, we quantified the pri-miRNAs and pre-miRNAs with absolute qRT-PCR based on SYBR Green I fluorescence. This improvement corrected for the inaccuracy caused by the differences in amplicon length and PCR efficiency. We also used SYBR Green method to quantify mature miRNAs based on the stem-loop qRT-PCR method. We extended the pairing part of the stem-loop reverse transcript (RT) primer from 6 to 11 bp, which greatly increased the efficiency of reverse transcription PCR (RT-PCR). The performance of the improved RT primer was tested using synthetic mature miRNAs and tissue RNA samples. Results showed that the improved RT primer demonstrated dynamic range of seven orders of magnitude and sensitivity of detection of hundreds of copies of miRNA molecules.
authors: The College of Environmental Science & Engineering, Donghua University, 2999 North Renmin Road, Songjiang, 200237, Shanghai, China.
authors: Li
authors: L
authors: Tong
authors: Huihui
authors: H
authors: Xue
authors: Li
authors: L
authors: Xiong
authors: Junhua
authors: J
authors: Xiao
authors: Yuxun
authors: Y
authors: Zhou
mesh_terms: D000818:Animals; D055029:Inverted Repeat Sequences; D051379:Mice; D035683:MicroRNAs / Q000032:analysis* / Q000737:chemistry; D012313:RNA / Q000235:genetics; D012322:RNA Precursors / Q000032:analysis / Q000737:chemistry; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D035683:MicroRNAs; D012322:RNA Precursors; C034477:RNA primers; D012313:RNA