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3′IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR: Impact on Small RNA-Seq Profiling Validation

Small RNA-sequencing (small RNA-seq) has revealed the presence of small RNA-naturally occurring variants such as microRNA (miRNA) isoforms or isomiRs. Due to their small size and the sequence similarity among miRNA isoforms, their validation by RT-qPCR is challenging. We previously identified two mi...

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Autores principales: Ferre, Adriana, Santiago, Lucía, Sánchez-Herrero, José Francisco, López-Rodrigo, Olga, Sánchez-Curbelo, Josvany, Sumoy, Lauro, Bassas, Lluís, Larriba, Sara
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10607168/
https://www.ncbi.nlm.nih.gov/pubmed/37895116
http://dx.doi.org/10.3390/ijms242015436
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author Ferre, Adriana
Santiago, Lucía
Sánchez-Herrero, José Francisco
López-Rodrigo, Olga
Sánchez-Curbelo, Josvany
Sumoy, Lauro
Bassas, Lluís
Larriba, Sara
author_facet Ferre, Adriana
Santiago, Lucía
Sánchez-Herrero, José Francisco
López-Rodrigo, Olga
Sánchez-Curbelo, Josvany
Sumoy, Lauro
Bassas, Lluís
Larriba, Sara
author_sort Ferre, Adriana
collection PubMed
description Small RNA-sequencing (small RNA-seq) has revealed the presence of small RNA-naturally occurring variants such as microRNA (miRNA) isoforms or isomiRs. Due to their small size and the sequence similarity among miRNA isoforms, their validation by RT-qPCR is challenging. We previously identified two miR-31-5p isomiRs—the canonical and a 3′isomiR variant (3′ G addition)—which were differentially expressed between individuals with azoospermia of different origin. Here, we sought to determine the discriminatory capacity between these two closely-related miRNA isoforms of three alternative poly(A) based-RT-qPCR strategies in both synthetic and real biological context. We found that these poly(A) RT-qPCR strategies exhibit a significant cross-reactivity between these miR-31-5p isomiRs which differ by a single nucleotide, compromising the reliable quantification of individual miRNA isoforms. Fortunately, in the biological context, given that the two miRNA variants show changes in the same direction, RT-qPCR results were consistent with the findings of small RNA-seq study. We suggest that miRNA selection for RT-qPCR validation should be performed with care, prioritizing those canonical miRNAs that, in small RNA-seq, show parallel/homogeneous expression behavior with their most prevalent isomiRs, to avoid confounding RT-qPCR-based results. This is suggested as the current best strategy for robust biomarker selection to develop clinically useful tests.
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spelling pubmed-106071682023-10-28 3′IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR: Impact on Small RNA-Seq Profiling Validation Ferre, Adriana Santiago, Lucía Sánchez-Herrero, José Francisco López-Rodrigo, Olga Sánchez-Curbelo, Josvany Sumoy, Lauro Bassas, Lluís Larriba, Sara Int J Mol Sci Article Small RNA-sequencing (small RNA-seq) has revealed the presence of small RNA-naturally occurring variants such as microRNA (miRNA) isoforms or isomiRs. Due to their small size and the sequence similarity among miRNA isoforms, their validation by RT-qPCR is challenging. We previously identified two miR-31-5p isomiRs—the canonical and a 3′isomiR variant (3′ G addition)—which were differentially expressed between individuals with azoospermia of different origin. Here, we sought to determine the discriminatory capacity between these two closely-related miRNA isoforms of three alternative poly(A) based-RT-qPCR strategies in both synthetic and real biological context. We found that these poly(A) RT-qPCR strategies exhibit a significant cross-reactivity between these miR-31-5p isomiRs which differ by a single nucleotide, compromising the reliable quantification of individual miRNA isoforms. Fortunately, in the biological context, given that the two miRNA variants show changes in the same direction, RT-qPCR results were consistent with the findings of small RNA-seq study. We suggest that miRNA selection for RT-qPCR validation should be performed with care, prioritizing those canonical miRNAs that, in small RNA-seq, show parallel/homogeneous expression behavior with their most prevalent isomiRs, to avoid confounding RT-qPCR-based results. This is suggested as the current best strategy for robust biomarker selection to develop clinically useful tests. MDPI 2023-10-21 /pmc/articles/PMC10607168/ /pubmed/37895116 http://dx.doi.org/10.3390/ijms242015436 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Ferre, Adriana
Santiago, Lucía
Sánchez-Herrero, José Francisco
López-Rodrigo, Olga
Sánchez-Curbelo, Josvany
Sumoy, Lauro
Bassas, Lluís
Larriba, Sara
3′IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR: Impact on Small RNA-Seq Profiling Validation
title 3′IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR: Impact on Small RNA-Seq Profiling Validation
title_full 3′IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR: Impact on Small RNA-Seq Profiling Validation
title_fullStr 3′IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR: Impact on Small RNA-Seq Profiling Validation
title_full_unstemmed 3′IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR: Impact on Small RNA-Seq Profiling Validation
title_short 3′IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR: Impact on Small RNA-Seq Profiling Validation
title_sort 3′isomir species composition affects reliable quantification of mirna/isomir variants by poly(a) rt-qpcr: impact on small rna-seq profiling validation
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10607168/
https://www.ncbi.nlm.nih.gov/pubmed/37895116
http://dx.doi.org/10.3390/ijms242015436
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