Technical analysis for detection and quantification of microRNAs by two real-time quantitative reverse transcription methods / 两种实时定量RT-PCR方法检测miRNAs表达的技术分析

• Published in: Xi'an jiao tong da xue xue bao. Journal of Xi'an Jiaotong University (medical sciences). Yi xue ban no. 2; p. 258
• Main Authors: Zi-xin, MIN, Xiao-yun, DU, Qi-lan, NING, Nan-nan, ZHONG, Yue-wen, ZHENG, HAN, Yan, She-min, Lü, ZHANG, Rui
• Format: Journal Article
• Language: Chinese
• Published: Xi'an Xi'an Jiaotong University 01.01.2013
• Subjects: Methods; MicroRNAs
• ISSN: 1671-8259
• DOI: 10.7652/jdyxb201302028

Objective: To optimize the method for quantifying microRNAs in different experimental purposes and conditions by comparing two real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) methods. Methods: We isolated the total RNA from the SD rat articular cartilage at postnatal day 21 and day 42 with TRIzol reagent. The RT reactions were performed by stem-loop primers and the universal primer of miRNA detection kit respectively; then real time PCR was performed to test the expressions of rno-miR-15b, rno-miR-16, rno-miR-195 and rno-miR-497. In addition, the total RNAs in human plasma were isolated by using TRI Reagent BD (MRC, TR126) according to the instructions by the manufacturer with two different RT-qPCR methods to quantify the expression of has-miR-16. Results: The expression change of these miRNAs was of the same increase trend by the two different RT-qPCR methods, which accorded with the results of our Solexa sequencing. The results of plasma demonstrated that stem-loop RT-qPCR m