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

Voss, Gjendine LU ; Edsjö, Anders LU ; Bjartell, Anders LU and Ceder, Yvonne LU orcid (2021) In RNA 27(11). p.1412-1424
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... (More)

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 as well as changes of editing levels in ADAR-overexpressing cell lines. 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.

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author
; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
RNA
volume
27
issue
11
pages
1412 - 1424
publisher
Cold Spring Harbor Laboratory Press (CSHL)
external identifiers
  • pmid:34433636
  • scopus:85118289662
ISSN
1355-8382
DOI
10.1261/rna.078867.121
language
English
LU publication?
yes
id
b147c41e-8ce3-47df-b6c9-fb4f1d252271
date added to LUP
2021-08-27 13:10:17
date last changed
2024-06-15 15:15:11
@article{b147c41e-8ce3-47df-b6c9-fb4f1d252271,
  abstract     = {{<p>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 as well as changes of editing levels in ADAR-overexpressing cell lines. 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.</p>}},
  author       = {{Voss, Gjendine and Edsjö, Anders and Bjartell, Anders and Ceder, Yvonne}},
  issn         = {{1355-8382}},
  language     = {{eng}},
  number       = {{11}},
  pages        = {{1412--1424}},
  publisher    = {{Cold Spring Harbor Laboratory Press (CSHL)}},
  series       = {{RNA}},
  title        = {{Quantification of microRNA editing using two-tailed RT-qPCR for improved biomarker discovery}},
  url          = {{http://dx.doi.org/10.1261/rna.078867.121}},
  doi          = {{10.1261/rna.078867.121}},
  volume       = {{27}},
  year         = {{2021}},
}