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

Rikki A M Brown,Michael R Epis,Jessica L Horsham,Tasnuva D Kabir,Peter J Leedman,Kirsty L Richardson

doi:10.1186/s12896-018-0421-6

Abstract

BackgroundmicroRNAs (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.ResultsWe 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.ConclusionsDifferent 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.

Highlights

MicroRNAs are short non-coding RNAs that fine-tune gene expression
If there is a limited amount of biological material from which to extract the RNA, researchers may be interested in the yield produced by different RNA extraction methods
Quantitation of the RNA using a Qubit Fluorometer revealed that the Bioline Isolate II kit consistently recovered lower amounts of RNA, for the brain and liver samples, and the miRNeasy kit consistently gave a high yield of RNA across all samples

Summary

Introduction

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. MicroRNAs (miRNAs) are a family of short (~ 22 nucleotide) non-coding RNAs that are essential for controlling the regulation of gene expression They act via binding to specific sequences in the 3′-untranslated region (3′-UTR) of mRNAs, leading to either. All rely on the quality of the in-put material Detection methods such as qRT-PCR and microarray are dependent on the assumption that the RNA extraction method isolates all miRNAs [7,8,9]. It is becoming evident that here is a crucial need to optimise and standardise the isolation of miRNA, given the discrepancies among many studies, whereby the recovery of miRNAs is dependent on the extraction method used [10,11,12,13,14,15,16]. These initial studies have focussed on miRNA recovery from plasma, urine and other bodily fluids, to date few papers have compared differences in miRNA extraction efficiency from solid tissues

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