View : 12 Download: 0

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

Title
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
Authors
Ban E.Kwon H.Seo H.S.Yoo Y.S.Song E.J.
Ewha Authors
송은주반은미
SCOPUS Author ID
송은주scopus; 반은미scopus
Issue Date
2021
Journal Title
BMC Biotechnology
ISSN
1472-6750JCR Link
Citation
BMC Biotechnology vol. 21, no. 1
Keywords
Amplification efficiencyBiomarkermiRNAPlasmaqRT-PCR
Publisher
BioMed Central Ltd
Indexed
SCIE; SCOPUS WOS scopus
Document Type
Article
Abstract
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. © 2021, The Author(s).
DOI
10.1186/s12896-021-00710-w
Appears in Collections:
약학대학 > 약학과 > Journal papers
Files in This Item:
There are no files associated with this item.
Export
RIS (EndNote)
XLS (Excel)
XML


qrcode

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

BROWSE