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

Eunmi Ban,Young Sook Yoo,Hong Seog Seo,Eun Joo Song,Haejin Kwon

doi:10.1186/s12896-021-00710-w

Abstract

BackgroundAlthough 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.MethodsThe 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.ResultsAn 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.ConclusionThis 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.

Highlights

Quantitative real-time PCR is a common and sensitive method for miRNAs analysis, it is necessary to optimize conditions and minimize quantitative real-time PCR (qRT-PCR) inhibitors to achieve reliable results
Evaluations of amplification efficiency are important for analyses of miRNAs in plasma because the miRNA concentrations are very low and the reaction can be affected by sample matrix and contaminants. This was a pilot study aimed at optimizing sample extraction and qRT-PCR conditions for reliable endogenous miRNA analyses in plasma
For the optimization of the annealing temperature for miRNA-16 and cel-miRNA-39, standard curves were constructed by plotting Quantification cycle (Cq) values against the logarithmic dilution factors using five and four tenfold serial dilutions of complementary DNA (cDNA) templet generated from synthetic miRNA-16 and Cel-miRNA-39-1, respectively as shown in Figs. 1 and 2

Summary

Introduction

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. In. Quantitative real-time PCR (qRT-PCR) is typically used to analyze miRNAs in the serum or plasma. CDNA amplification by PCR is often restricted by various issues, such as an ineffective annealing temperature or the presence of PCR inhibitors [6]. These factors can dramatically reduce the sensitivity and amplification efficiency of PCR, leading to the misinterpretation of results and a low reproducibility [7]

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