Multicenter evaluation of miRNA extraction technologies for the development of a clinically relevant miRNA analysis workflow

MicroRNAs (miRNAs) are small noncoding RNAs that interfere with and regulate different biological pathways. Therefore, dysregulation of miRNAs could lead to disease initiation and progression, particularly in various types of cancer. In human body fluids, miRNAs can be found as circulating cell-free miRNAs (cfmiRNAs, often associated with RNA-binding proteins) as well as exosomal miRNAs (exomiRNAs), both serving as potentially valuable biomarkers to evaluate patient’s disease progression, or to monitor treatment efficacy. Various methods for the extraction and analysis of miRNAs, cfmiRNAs or exomiRNAs, are commercially available. Here, we describe the efforts of the Innovative Medicines Initiative (IMI) consortium CANCER-ID (www.cancer-id.eu), a public-private partnership of experts from academia, biotech and pharmaceutical industry, in generating comparative data using different RNA/miRNA extraction technologies in a multicenter ring study.

To address clinical relevance in a later stage of the project, K2EDTA plasma samples from 20 healthy donors were collected in a centralized way with respective informed consent. Five different RNA/miRNA extraction methods and two extraction methods specific for exomiRNAs were selected for comparison: mirVana (ThermoFisher Scientific), miRCURY RNA Isolation Kit (Exiqon), miRNeasy Serum/Plasma kit (QIAGEN), miRNeasy Advanced Serum/Plasma kit (QIAGEN), Plasma/Serum RNA Purification mini kit (Norgen), exoRNeasy Serum/Plasma (QIAGEN) and ultracentrifugation. The eluates were sent to one CANCER-ID partner, centrally performing RT-qPCR analysis on six miRNAs, as well as miRNA massive parallel sequencing.

After extraction using the miRCURY RNA isolation kit and miRNeasy Advanced Serum/Plasma kit, highest miRNA-seq calls (mean: 2×10^6) and lowest RT-qPCR Cq-values (mean: 26.6) for six miRNAs were observed with both extraction methods. Samples from patients and healthy volunteers, respectively, were collected under signed informed consent.

Based on the results of the evaluation of the different miRNA extraction technologies a single extraction technology will be selected for the next iteration of ring studies, the comparison of different analysis platforms and the integration of downstream workflows. First results of these ring studies will be presented.

Martin H. D. Neumann (Bayer AG), Francesca Di Pasquale (QIAGEN), Markus Sprenger-Haussels (QIAGEN), Jonathan M. Shaffer (QIAGEN Sciences), Martin Schlumpberger (QIAGEN), Fay Betsou (IBBL) Wim Ammerlaan (IBBL), Taija af Hallström (Orion Oyi), Evi Lianidou (University of Athens), Robert Sjöback (TATAA Biocenter), Sebastian Bender (Bayer), Thomas Schlange (Bayer)

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