Comparison of Two Available RNA Extraction Protocols for MicroRNA Amplification in Serum Samples

Overview

Journal J Clin Lab Anal

Publisher Wiley

Specialties Biomedical Engineering
Biotechnology
Pathology

Date 2015 Apr 9

PMID 25853736

Citations 8

Authors

Massimiliano Bergallo

Stefano Gambarino

Silvana Martino

Davide Montin

Paola Montanari

Ilaria Galliano

Pier-Angelo Tovo

Affiliations

Soon will be listed here.

Abstract

Background: microRNAs play a critical role in many biological processes such as cell proliferation and maturation, apoptosis, regulation of chronic inflammation and development of cancer.

Methods: In this study is described a protocol for the isolation of RNA from serum and subsequent determination of miRNA expression levels using TaqMan-based MGB Real-Time PCR detection. RNA was extracted using two different isolation methods including available kits RNAzol and a modified RNAzol protocol. In all cases, RNA was eluted in RNase free H2 O, kept frozen until analysis and the presence of contaminants assessed by NanoDrop spectrophotometry.

Results: Higher RNA quantity was observed in RNAzol (378.8 ng/μl) vs RNAzol modified protocol (226.5 ng/μl) and a better performance in terms of RNA extraction yield and purity. Subsequently, measurements of endogenous miRNAs (RNU43), cellular miRNAs (mir155 and mir146a) and EBV miRNAs (mirBART2-5p, mirBART15 and mirBART22) were performed by RT-qPCR.

Conclusion: In contrast to the findings in terms of purity and quantity, the amplifiable RNA was more abundant using RNAzol modified protocol compared to not modified protocol.

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Rodriguez A, Duyvejonck H, van Belleghem J, Gryp T, Van Simaey L, Vermeulen S PLoS One. 2020; 15(2):e0229423.

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