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

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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 H2O, 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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