Open AccessComparison of Two Available RNA Extraction Protocols for microRNA Amplification in Serum Samples
Author(s) -
Bergallo Massimiliano,
Gambarino Stefano,
Martino Silvana,
Montin Davide,
Montanari Paola,
Galliano Ilaria,
Tovo PierAngelo
Publication year - 2016
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.21848
Subject(s) - rna , rna extraction , taqman , microrna , rnase p , microbiology and biotechnology , real time polymerase chain reaction , biology , nuclease protection assay , nucleic acid , chemistry , chromatography , non coding rna , biochemistry , gene
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 H 2 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.