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RNA. 2019 Feb;25(2):232-238. doi: 10.1261/rna.069047.118. Epub 2018 Nov 28.

miRNA length variation during macrophage stimulation confounds the interpretation of results: implications for miRNA quantification by RT-qPCR.

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Centre for Cancer Biology, SA Pathology and University of South Australia, Adelaide, South Australia 5000, Australia.
ACRF Cancer Genomics Facility, Centre for Cancer Biology, SA Pathology, Adelaide, South Australia 5000, Australia.
School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia 5005, Australia.
Department of Medicine, University of Adelaide, Adelaide, South Australia 5005, Australia.
Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Victoria 3168, Australia.
Department of Molecular and Translational Science, Monash University, Clayton, Victoria 3168, Australia.


Most microRNAs (miRNAs) are expressed as a mix of length isoforms (referred to as isomiRs). IsomiR stoichiometry can be differentially impacted upon cell stimulation, as recently evidenced by our group in the context of immune responses induced by type-I interferon (IFN). Here, we revisit published RNA-seq data sets of human and mouse macrophages stimulated with bacterial products at the isomiR level. We demonstrate that for several miRNAs, macrophage stimulation induces changes in isomiR stoichiometry. Critically, we find that changes in miRNA expression can be misinterpreted when miRNAs are quantified by RT-qPCR, as primers directed against canonical miRNA sequences may not equally target the different isomiRs that are regulated endogenously. Beyond the case of phagocyte stimulation, our analyses reinforce the concept that analysis of miRNA expression at the isoform level should become standard practice.


LPS; RT-qPCR miRNA assays; bacterial infection; interferon; isomiR; macrophages; microRNA isoform


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