Abstract 5043: Comparison of the efficacy and analytical performance of isolation procedures for circulating plasma microRNAs

Abstract

Abstract Introduction: The efficiency and reproducibility of plasma miRNAs extraction protocol consists a critical step concerning the quantification of circulating miRNAs in plasma. Plasma is a specimen with high concentrations of lipids and proteins, so specific handling is required in order to achieve an enriched fraction of small RNAs at the end of the procedure. In the present study, we conducted a comparative investigation regarding RNA yielding among different commercially available experimental approaches. Our evaluation approach relied on the comparative extraction yielding of endogenous human miR-21 and exogenous spiked-in C.elegans miR-39 from pooled plasma samples from healthy individuals. Materials and Methods: In the present study we evaluated the extraction potential of small RNAs using three different commercially available kits for miRNAs extraction from plasma samples according to the manufacturer's protocols: a) miRNeasy mini Kit (Qiagen), b) mirVana PARIS kit (Ambion), c) microRNA purification Kit (Norgen Biotek) and d) Trizol LS Reagent (Invitrogen). We firstly pooled plasma samples from healthy individuals and used them for spiking experiments. In all cases, the initial plasma volume was 200μL and in each sample we spiked 25fmol of the exogenous standard C.elegans cel-miR-39. The isolation of miRNAs from plasma was conducted as per kit protocols and all extraction methods were performed three independent times from the same initial pooled plasma sample. A fixed volume of the eluted RNA sample was used as an input for the reverse transcription reaction. After isolation, quantification of cel-miR-39 was performed using real-time PCR. Recovery of cel-miR-39 in each case was estimated in respect to the levels of an equivalent amount of C.elegans copies added to total RNA after each extraction method (representing 100% recovery). In parallel, in all these samples the endogenous hsa-miR-21 was also quantified by real-time qPCR. The results were compared between the different isolation protocols concerning this with the greatest miRNAs yield for expression levels of endogenous miRNAs. Results: We found that the phenol:chloroform extraction and ethanol precipitation of RNA protocol (Trizol LS) recovers small RNAs with some loss whereas silica-column based RNA extraction methods provide a more effective and reliable mechanism for the isolation of small RNAs. Although all column protocols have indications to be very effective, in our hands the mirVana PARIS (Ambion) protocol appeared giving higher yields of circulating miRNAs from plasma samples. This was the case after quantifying for both the exogenous (cel-miR-39) and endogenous (miR-21) less abundant levels of miRNAs. Conclusion: According to our results, the mirVana PARIS kit (Ambion) was the most efficient among different commercially available kits for the isolation of circulating plasma miRNAs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5043. doi:1538-7445.AM2012-5043