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Systematic comparison of microarray profiling, real-time PCR, and nextgeneration sequencing technologies for measuring differential microRNA expression

Systematic comparison of microarray profiling, real-time PCR, and nextgeneration sequencing technologies for measuring differential microRNA expression

Anna Git1,3, Heidi Dvinge2,3, Mali Salmon-Divon2,3, Michelle Osborne1, Claudia Kutter1, James Hadfield1, Paul Bertone2 and Carlos Caldas1
1. Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Cambridge CB2 0RE, United Kingdom
2. European Molecular Biology Laboratory–European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Cambridge CB10 1SD, United Kingdom
3. These authors contributed equally to this work

Abstract

RNA abundance and DNA copy number are routinely measured in high-throughput using microarray and next-generation sequencing (NGS) technologies, and the attributes of different platforms have been extensively analyzed. Recently, the application of both microarrays and NGS has expanded to include microRNAs (miRNAs), but the relative performance of these methods has not been rigorously characterized. We analyzed three biological samples across six miRNA microarray platforms and compared their hybridization performance. We examined the utility of these platforms, as well as NGS, for the detection of differentially expressed miRNAs. We then validated the results for 89 miRNAs by real-time RT-PCR and challenged the use of this assay as a “gold standard.” Finally, we implemented a novel method to evaluate false-positive and false-negative rates for all methods in the absence of a reference method

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