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

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.Electronic supplementary materialThe online version of this article (10.1186/s12896-018-0421-6) contains supplementary material, which is available to authorized users.

Challenges in Using Circulating Micro-RNAs as Biomarkers for Cardiovascular Diseases

“…However, it is still difficult to quantify the levels of c-miRNAs due to their low concentrations while all the miRNA detection methods depend on the quality of the starting material. In addition, the most widely used methods including qRT-PCR and microarray are based on the assumption that the different RNA extraction methods isolate all miRNAs equally [29]. However, as discussed above the levels of c-miRNA isolated using different methods should not be directly compared as the recovery of c-miRNA varies dramatically [127,128].…”

“…Additionally, due to their low concentration in plasma and serum, miRNAs can only be quantified using amplification techniques and thus the amount of starting material and the extraction method may affect the results [28]. It has been demonstrated that sample type and processing, as well as RNA extraction methods significantly affect the miRNA profile and/or expression levels [28,29]. Importantly, a comparison of c-miRNA profiles obtained by different laboratories for the same disease, including CVDs, revealed different expression levels of specific miRNAs (see [30] and references cited therein), illustrating the need to standardize the detection and analysis of c-miRNAs.…”

MicroRNA‑138 inhibits SOX12 expression and the proliferation, invasion and migration of ovarian cancer cells

“…Then, total RNA was extracted using the phenol chloroform method (21). The purity of RNA was determined by A260/A280 using ultraviolet spectrophotometry (Nanodrop 2000 Spectrophotometer; Thermo Fisher Scientific, Inc.).…”

Comprehensive Profiling of the Circulatory miRNAome Response to a High Protein Diet in Elderly Men: A Potential Role in Inflammatory Response Modulation

“…However no correlations between fold changes in sequencing reads and qPCR abundance for expression of miR‐100‐5p, ‐23b‐5p, and ‐203a were detected. This observed discordance again reflects the uncertainties imposed by different RNA isolation methods, in this study to undertake qPCR analysis, ‐phenol chloroform‐based extraction method was used, differing from the solid‐phase separation with affinity resin based extraction that is necessary for purifying better quality RNA needed for HTS . Further, quantification of miRNA expression by qPCR is influenced by the choice of reference genes used for normalization .…”