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.

Keywords: Biomarkers; Extraction; Real-time PCR; Tissue; miRNA-based therapy; microRNA isolation method; microRNAs.

Fig. 1

Experimental design for comparing RNA isolation methods from mouse tissues. Brain, lung and liver tissues were dissected from C57BL/6 mice (n = 3) and rapidly frozen. Tissues were homogenised and RNA extracted using five different methods and assessed for yield, purity, integrity and miRNA abundance. miRNA and target gene expression was then quantitated with qRT-PCR

Fig. 2

Flow chart summarising differences in the protocols, time requirements and prices of the different RNA purification methods. RNA was isolated using five methods that were either phenol based (TRIzol), column-based (Isolate II and Norgen total), or combined phenol and column-based methods (miRNeasy and miRVana). $ < 5, $$ = 5–10, $$$ > 10 AUD$ per sample preparation. Time requirement based on following instructions provided by manufacturer (not taking into account extra time required by the technician to perform the tasks)

Fig. 3

Quantitative real-time PCR analyses of 6 miRNAs comparing different RNA extraction methods. RNA was extracted from murine brain (a), liver (b) and lung (c) tissues using the five indicated methods and endogenous miRNA expression quantitated using TaqMan MicroRNA Assays via qRT-PCR. miRNA expression was normalised using sno202 as a reference gene. Values indicate the average normalised miRNA expression (technical duplicates from triplicate biological isolations) in different tissues on a Log2 scale with SEM error bars. * p < 0.05 when compared to three or more other methods

Fig. 4

Quantitative real-time PCR analyses comparing miRNA expression patterns in tissues using different extraction methods. Fold abundance of miRNAs quantitated using TaqMan MicroRNA Assays via qRT-PCR from brain (a), liver (b) and lung (c) tissues. miRNA expression was normalised using sno202 as a reference gene. Values indicate the average normalised miRNA expression in different tissues on a Log2 scale with SEM error bars. * p < 0.05 when compared to five the other miRNAs for each method

Fig. 5

Quantitative real-time PCR analyses of target gene expression from murine tissues. EGFR and AXL expression was analysed from the same RNA samples isolated from brain (a), liver (b) and lung (c) tissues for miRNA detection. Target gene expression was normalised to either: B2M and 18S/HPRT as reference genes. Values indicate the average normalised gene expression (technical duplicates from triplicate isolations) in different tissues on a Log2 scale with SEM error bars. * p < 0.05 when compared to three or more other methods

Fig. 6

Quantitative real-time PCR analyses of relative house-keeping gene expression in the various murine tissues. Sno202 (a), B2M (b), HPRT (c) and 18S (d) expression was assessed via RT-qPCR and GeNorm and Normfinder used to determine suitability for normalisation. Values indicated the average gene expression (technical duplicates from triplicate isolations) with SEM error bars.* p < 0.05 when compared to three or more other methods