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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Detalles Bibliográficos
Autores: Ferré, Adriana, Santiago, Lucía, Sánchez Herrero, José Francisco, López Rodrigo, Olga, Sánchez Curbelo, Josvany, Sumoy, Lauro, Bassas, Lluís, Larriba, Sara
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2023
País:España
Institución:Universidad de Barcelona
Repositorio:Dipòsit Digital de la UB
OAI Identifier:oai:diposit.ub.edu:2445/209210
Acceso en línea:https://hdl.handle.net/2445/209210
Access Level:acceso abierto
Palabra clave:Micro RNAs
Càncer
MicroRNAs
Cancer
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spelling 3′IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR: Impact on Small RNA-Seq Profiling ValidationFerré, AdrianaSantiago, LucíaSánchez Herrero, José FranciscoLópez Rodrigo, OlgaSánchez Curbelo, JosvanySumoy, LauroBassas, LluísLarriba, SaraMicro RNAsCàncerMicroRNAsCancerSmall 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 AG2023info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://hdl.handle.net/2445/209210Articles publicats en revistes (Institut d'lnvestigació Biomèdica de Bellvitge (IDIBELL))reponame:Dipòsit Digital de la UBinstname:Universidad de BarcelonaInglésReproducció del document publicat a: https://doi.org/10.3390/ijms242015436International Journal of Molecular Sciences, 2023, vol. 24, num. 20https://doi.org/10.3390/ijms242015436cc by (c) Ferré, Adriana et al., 2023http://creativecommons.org/licenses/by/3.0/es/info:eu-repo/semantics/openAccessoai:diposit.ub.edu:2445/2092102026-05-27T06:46:51Z
dc.title.none.fl_str_mv 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
spellingShingle 3′IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR: Impact on Small RNA-Seq Profiling Validation
Ferré, Adriana
Micro RNAs
Càncer
MicroRNAs
Cancer
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_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_sort 3′IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR: Impact on Small RNA-Seq Profiling Validation
dc.creator.none.fl_str_mv Ferré, Adriana
Santiago, Lucía
Sánchez Herrero, José Francisco
López Rodrigo, Olga
Sánchez Curbelo, Josvany
Sumoy, Lauro
Bassas, Lluís
Larriba, Sara
author Ferré, Adriana
author_facet Ferré, Adriana
Santiago, Lucía
Sánchez Herrero, José Francisco
López Rodrigo, Olga
Sánchez Curbelo, Josvany
Sumoy, Lauro
Bassas, Lluís
Larriba, Sara
author_role author
author2 Santiago, Lucía
Sánchez Herrero, José Francisco
López Rodrigo, Olga
Sánchez Curbelo, Josvany
Sumoy, Lauro
Bassas, Lluís
Larriba, Sara
author2_role author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Micro RNAs
Càncer
MicroRNAs
Cancer
topic Micro RNAs
Càncer
MicroRNAs
Cancer
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.
publishDate 2023
dc.date.none.fl_str_mv 2023
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv https://hdl.handle.net/2445/209210
url https://hdl.handle.net/2445/209210
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv Reproducció del document publicat a: https://doi.org/10.3390/ijms242015436
International Journal of Molecular Sciences, 2023, vol. 24, num. 20
https://doi.org/10.3390/ijms242015436
dc.rights.none.fl_str_mv cc by (c) Ferré, Adriana et al., 2023
http://creativecommons.org/licenses/by/3.0/es/
info:eu-repo/semantics/openAccess
rights_invalid_str_mv cc by (c) Ferré, Adriana et al., 2023
http://creativecommons.org/licenses/by/3.0/es/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv MDPI AG
publisher.none.fl_str_mv MDPI AG
dc.source.none.fl_str_mv Articles publicats en revistes (Institut d'lnvestigació Biomèdica de Bellvitge (IDIBELL))
reponame:Dipòsit Digital de la UB
instname:Universidad de Barcelona
instname_str Universidad de Barcelona
reponame_str Dipòsit Digital de la UB
collection Dipòsit Digital de la UB
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,300724

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