Background: Although quantitative real-time PCR (qRT-PCR) is a common and sensitive method for miRNAs analysis, it is necessary to optimize conditions and minimize qRT-PCR inhibitors to achieve reliable results. The aim of this study was to minimize interference by contaminants in qRT-PCR, maximize product yields for miRNA analyses, and optimize PCR conditions for the reliable screening of miRNAs in plasma. Methods: The annealing temperature was first optimized by assessing amplification efficiencies. The effects of extraction conditions on levels of inhibitors that interfere with PCR were evaluated. The tested extraction conditions were the volume of the upper layer taken, number of chloroform extractions, and the inclusion of ethanol washing, a process that reduces PCR interference during RNA extraction using TRIzol. Results: An acceptable amplification efficiency of RT-qPCR was achieved by the optimization of the annealing temperature of the tested miRNAs and by the collection a supernatant volume corresponding to about 50% of the volume of TRIzol with triple chloroform extraction. These optimal extraction and PCR conditions were successfully applied to plasma miRNA screening to detect biomarker candidates for the diagnosis of acute myocardial infarction. Conclusion: This is the first study to optimize extraction and qRT-PCR conditions, while improving miRNA yields and minimizing the loss of extracted miRNA by evaluations of the amplification efficiency.
Bibliographical noteFunding Information:
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (2020R1A4A4079494 and 2019R1A2C2004052), Korea Basic Science Institute (National research Facilities and Equipment Center) grant funded by the Ministry of Education (2021R1A6C101A442) and intramural grant from Korea Institute of Science and Technology (2E30441). The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
© 2021, The Author(s).
- Amplification efficiency