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bioRxiv · 10.1101/2021.06.15.448409

Quantification of microRNA editing using two-tailed RT-qPCR for improved biomarker discovery

Voss, G.·Edsjö, A.·Bjartell, A.·Ceder, Y.
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Abstract

Even though microRNAs have been viewed as promising biomarkers for years, their clinical implementation is still lagging far behind. This is in part due to the lack of RT-qPCR technologies that can differentiate between microRNA isoforms. For example, A-to-I editing of microRNAs through adenosine deaminase acting on RNA (ADAR) enzymes can affect their expression levels and functional roles, but editing isoform-specific assays are not commercially available. Here, we describe RT-qPCR assays that are specific for editing isoforms, using microRNA-379 (miR-379) as a model. The assays are based on two-tailed RT-qPCR, and we show them to be compatible both with SYBR Green and hydrolysis-based chemistries, as well as with both qPCR and digital PCR. The assays could readily detect different miR-379 editing isoforms in various human tissues. We found that the miR-379 editing frequency was higher in prostate cancer samples compared to benign prostatic hyperplasia samples. Furthermore, decreased expression of unedited miR-379, but not edited miR-379, was associated with treatment resistance, metastasis and shorter overall survival. Taken together, this study presents the first RT-qPCR assays that were demonstrated to distinguish A-to-I-edited microRNAs, and shows that they can be useful in the identification of biomarkers that previously have been masked by other isoforms. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/448409v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@79afcorg.highwire.dtl.DTLVardef@15028deorg.highwire.dtl.DTLVardef@1ab3fe9org.highwire.dtl.DTLVardef@1f11760_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Voss, G., Edsjö, A., Bjartell, A., Ceder, Y.. 2021-06-15. Quantification of microRNA editing using two-tailed RT-qPCR for improved biomarker discovery. https://doi.org/10.1101/2021.06.15.448409

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