Ban, Eunmi Kwon, Haejin Seo, Hong Seog Yoo, Young Sook Song, Eun Joo 2024-01-19T14:02:44Z 2024-01-19T14:02:44Z 2022-01-25 2021-08 1472-6750 https://pubs.kist.re.kr/handle/201004/116615 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. English BMC Screening of miRNAs in plasma as a diagnostic biomarker for cardiac disease based on optimization of extraction and qRT-PCR condition assay through amplification efficiency Article 10.1186/s12896-021-00710-w 1 BMC BIOTECHNOLOGY, v.21, no.1 BMC BIOTECHNOLOGY 21 1 N scie scopus 000686675000002 2-s2.0-85112501447 Biotechnology & Applied Microbiology Biotechnology & Applied Microbiology Article ACUTE MYOCARDIAL-INFARCTION CIRCULATING MIRNAS MICRORNAS SERUM qRT-PCR miRNA Amplification efficiency Biomarker Plasma