Evaluation of the efficacy of various reagents in improving microRNA extraction.

Abstract

MicroRNA has received considerable attention in the clinical context, and attempts are being made to use microRNA in clinical diagnosis. However, adequate quantities of microRNA required for analysis are challenging to isolate. We tested the effect of various reagents in improving microRNA extraction and compared their efficacy to that of a commercially available extraction kit (HighPure miRNA isolation kit, Roche). We used the synthetic oligonucleotide miR-21 and formalin-fixed, paraffin-embedded (FFPE) tissue sections from colon cancer samples ( n = 10). We tested increasing volumes (100-600 μL) of 1,4-dioxane, 2-butanol, 2-propanol, acetonitrile, polyethylene glycol (PEG) 600, PEG 1000, PEG 1540, PEG 2000, tetraethylene glycol dimethyl ether (TDE), and tetrahydrofuran, instead of the binding enhancer solution provided in the kit. MiR-21 analysis was performed via stem-loop RT-qPCR using Universal ProbeLibrary probe (Roche). The optimum amount of each enhancement solution was 200-500 μL. We obtained ΔCp values of optimum additional volume for each solution from 1.04 to 2.50 and compared these with those obtained using the commercially available kit. PEG 1540 and 2000 produced superior reactivity with minimal addition. For FFPE tissue samples, addition of the enhancement solutions PEG 1540 and 2000 resulted in mean crossing point values of 18.15 ± 2.26 and 17.73 ± 3.26, respectively. We obtained a crossing point value of 20.56 ± 4.26 (mean ± SD) using the commercially available kit. The tested enhancer reagents, which are relatively readily available and easy to use, can improve microRNA extraction efficacy of a commercially available kit.