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Total RNA extraction from tissues for microRNA and target gene expression analysis: not all kits are created equal.

Abstract：

BACKGROUND:microRNAs (miRNAs) are short non-coding RNAs that fine-tune gene expression. The aberrant expression of miRNAs is associated with many diseases and they have both therapeutic and biomarker potential. However, our understanding of their usefulness is dependent on the tools we have to study them. Previous studies have identified the need to optimise and standardise RNA extraction methods in order to avoid biased results. Herein, we extracted RNA from murine lung, liver and brain tissues using five commercially available total RNA extraction methods. These included either: phenol: chloroform extraction followed by alcohol precipitation (TRIzol), phenol:chloroform followed by solid-phase extraction (column-based; miRVana and miRNeasy) and solid-phase separation with/without affinity resin (Norgen total and Isolate II). We then evaluated each extraction method for the quality and quantity of RNA recovered, and the expression of miRNAs and target genes. RESULTS:We identified differences between each of the RNA extraction methods in the quantity and quality of RNA samples, and in the analysis of miRNA and target gene expression. For the purposes of consistency in quantity, quality and high recovery of miRNAs from tissues, we identified that Phenol:chloroform phase separation combined with silica column-based solid extraction method was preferable (miRVana microRNA isolation). We also identified a method that is not appropriate for miRNA analysis from tissue samples (Bioline Isolate II). For target gene expression any of the kits could be used to analyse mRNA, but if interested in analysing mRNA and miRNA from the same RNA samples some methods should be avoided. CONCLUSIONS:Different methods used to isolate miRNAs will yield different results and therefore a robust RNA isolation method is required for reproducibility. Researchers should optimise these methods for their specific application and keep in mind that "total RNA" extraction methods do not isolate all types of RNA equally.

journal_name

BMC Biotechnol

journal_title

BMC biotechnology

authors

Brown RAM,Epis MR,Horsham JL,Kabir TD,Richardson KL,Leedman PJ

doi

10.1186/s12896-018-0421-6

subject

Has Abstract

pub_date

2018-03-16 00:00:00

pages

issue

issn

1472-6750

pii

10.1186/s12896-018-0421-6

journal_volume

pub_type

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Total RNA extraction from tissues for microRNA and target gene expression analysis: not all kits are created equal.