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Two-Tailed RT-qPCR for the Quantification of A-to-I-Edited microRNA Isoforms

Voss, Gjendine LU and Ceder, Yvonne LU orcid (2023) In Current protocols 3(1).
Abstract

MicroRNAs are short non-coding RNAs with important functions in the regulation of gene expression in healthy and diseased tissues. To optimally utilize the biological and clinical information that is contained in microRNA expression levels, tools for their accurate and cost-effective quantification are needed. While the standard method, qPCR, allows for quick and cheap microRNA quantification, specificity is limited due to the short lengths of microRNAs and the high similarity between closely related microRNA family members. A-to-I editing can further diversify the microRNA pool by altering individual nucleotides. There is currently a lack of protocols for the accurate quantification of A-to-I-edited microRNA isoforms using qPCR. Here,... (More)

MicroRNAs are short non-coding RNAs with important functions in the regulation of gene expression in healthy and diseased tissues. To optimally utilize the biological and clinical information that is contained in microRNA expression levels, tools for their accurate and cost-effective quantification are needed. While the standard method, qPCR, allows for quick and cheap microRNA quantification, specificity is limited due to the short lengths of microRNAs and the high similarity between closely related microRNA family members. A-to-I editing can further diversify the microRNA pool by altering individual nucleotides. There is currently a lack of protocols for the accurate quantification of A-to-I-edited microRNA isoforms using qPCR. Here, we describe a protocol to quantify microRNA editing isoforms using two-tailed RT-qPCR, with either SYBR Green or hydrolysis probes. The user will perform reverse transcription of RNA samples, generate standard curves, and quantify the resulting cDNA in the following qPCR step. We also give guidelines for primer design and for the evaluation of assays using synthetic oligonucleotides. These tools are expected to be transferable to any A-to-I-edited microRNA and its isoforms.

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author
and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
A-to-I editing, biomarker discovery, microRNA, non-coding RNAs, RNA editing, two-tailed RT-qPCR
in
Current protocols
volume
3
issue
1
article number
e645
publisher
John Wiley & Sons Inc.
external identifiers
  • scopus:85146621653
  • pmid:36688607
ISSN
2691-1299
DOI
10.1002/cpz1.645
language
English
LU publication?
yes
id
f4373fa7-9c81-4706-ac77-cc457e56ba24
date added to LUP
2023-02-14 09:33:42
date last changed
2024-04-18 18:44:20
@article{f4373fa7-9c81-4706-ac77-cc457e56ba24,
  abstract     = {{<p>MicroRNAs are short non-coding RNAs with important functions in the regulation of gene expression in healthy and diseased tissues. To optimally utilize the biological and clinical information that is contained in microRNA expression levels, tools for their accurate and cost-effective quantification are needed. While the standard method, qPCR, allows for quick and cheap microRNA quantification, specificity is limited due to the short lengths of microRNAs and the high similarity between closely related microRNA family members. A-to-I editing can further diversify the microRNA pool by altering individual nucleotides. There is currently a lack of protocols for the accurate quantification of A-to-I-edited microRNA isoforms using qPCR. Here, we describe a protocol to quantify microRNA editing isoforms using two-tailed RT-qPCR, with either SYBR Green or hydrolysis probes. The user will perform reverse transcription of RNA samples, generate standard curves, and quantify the resulting cDNA in the following qPCR step. We also give guidelines for primer design and for the evaluation of assays using synthetic oligonucleotides. These tools are expected to be transferable to any A-to-I-edited microRNA and its isoforms.</p>}},
  author       = {{Voss, Gjendine and Ceder, Yvonne}},
  issn         = {{2691-1299}},
  keywords     = {{A-to-I editing; biomarker discovery; microRNA; non-coding RNAs; RNA editing; two-tailed RT-qPCR}},
  language     = {{eng}},
  number       = {{1}},
  publisher    = {{John Wiley & Sons Inc.}},
  series       = {{Current protocols}},
  title        = {{Two-Tailed RT-qPCR for the Quantification of A-to-I-Edited microRNA Isoforms}},
  url          = {{http://dx.doi.org/10.1002/cpz1.645}},
  doi          = {{10.1002/cpz1.645}},
  volume       = {{3}},
  year         = {{2023}},
}