3'IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR

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: Ferre-Giraldo, Adriana, Santiago, Lucía, Sánchez Herrero, José Francisco|||0000-0001-6771-4807, López-Rodrigo, Olga|||0000-0001-9325-5835, Sánchez-Curbelo, Josvany|||0000-0002-6489-3309, Sumoy, Lauro|||0000-0003-0005-4618, Bassas, Lluís|||0000-0002-3473-3611, Larriba, Sara|||0000-0003-4579-5452
Tipo de recurso: artículo
Fecha de publicación:2023
País:España
Institución:Universitat Autònoma de Barcelona
Repositorio:Dipòsit Digital de Documents de la UAB
Idioma:inglés
OAI Identifier:oai:ddd.uab.cat:300101
Acceso en línea:https://ddd.uab.cat/record/300101
https://dx.doi.org/urn:doi:10.3390/ijms242015436
Access Level:acceso abierto
Palabra clave:IsomiRs
MiRNA isoforms
Poly(A)-RT-qPCR
Selective amplification
Small RNAseq validation strategy
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spelling 3'IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCRImpact on Small RNA-Seq Profiling ValidationFerre-Giraldo, AdrianaSantiago, LucíaSánchez Herrero, José Francisco|||0000-0001-6771-4807López-Rodrigo, Olga|||0000-0001-9325-5835Sánchez-Curbelo, Josvany|||0000-0002-6489-3309Sumoy, Lauro|||0000-0003-0005-4618Bassas, Lluís|||0000-0002-3473-3611Larriba, Sara|||0000-0003-4579-5452IsomiRsMiRNA isoformsPoly(A)-RT-qPCRSelective amplificationSmall RNAseq validation strategySmall 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.Universitat Autònoma de Barcelona 22023-01-0120232023-01-01Articlehttp://purl.org/coar/resource_type/c_6501VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfhttps://ddd.uab.cat/record/300101https://dx.doi.org/urn:doi:10.3390/ijms242015436reponame:Dipòsit Digital de Documents de la UABinstname:Universitat Autònoma de BarcelonaInglésengInstituto de Salud Carlos III https://doi.org/10.13039/501100004587 DTS18/00101open accesshttp://purl.org/coar/access_right/c_abf2Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.https://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:ddd.uab.cat:3001012026-06-06T12:50:31Z
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
spellingShingle 3'IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR
Ferre-Giraldo, Adriana
IsomiRs
MiRNA isoforms
Poly(A)-RT-qPCR
Selective amplification
Small RNAseq validation strategy
title_short 3'IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR
title_full 3'IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR
title_fullStr 3'IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR
title_full_unstemmed 3'IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR
title_sort 3'IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR
dc.creator.none.fl_str_mv Ferre-Giraldo, Adriana
Santiago, Lucía
Sánchez Herrero, José Francisco|||0000-0001-6771-4807
López-Rodrigo, Olga|||0000-0001-9325-5835
Sánchez-Curbelo, Josvany|||0000-0002-6489-3309
Sumoy, Lauro|||0000-0003-0005-4618
Bassas, Lluís|||0000-0002-3473-3611
Larriba, Sara|||0000-0003-4579-5452
author Ferre-Giraldo, Adriana
author_facet Ferre-Giraldo, Adriana
Santiago, Lucía
Sánchez Herrero, José Francisco|||0000-0001-6771-4807
López-Rodrigo, Olga|||0000-0001-9325-5835
Sánchez-Curbelo, Josvany|||0000-0002-6489-3309
Sumoy, Lauro|||0000-0003-0005-4618
Bassas, Lluís|||0000-0002-3473-3611
Larriba, Sara|||0000-0003-4579-5452
author_role author
author2 Santiago, Lucía
Sánchez Herrero, José Francisco|||0000-0001-6771-4807
López-Rodrigo, Olga|||0000-0001-9325-5835
Sánchez-Curbelo, Josvany|||0000-0002-6489-3309
Sumoy, Lauro|||0000-0003-0005-4618
Bassas, Lluís|||0000-0002-3473-3611
Larriba, Sara|||0000-0003-4579-5452
author2_role author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv Universitat Autònoma de Barcelona
dc.subject.none.fl_str_mv IsomiRs
MiRNA isoforms
Poly(A)-RT-qPCR
Selective amplification
Small RNAseq validation strategy
topic IsomiRs
MiRNA isoforms
Poly(A)-RT-qPCR
Selective amplification
Small RNAseq validation strategy
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 2
2023-01-01
2023
2023-01-01
dc.type.none.fl_str_mv Article
http://purl.org/coar/resource_type/c_6501
VoR
http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv https://ddd.uab.cat/record/300101
https://dx.doi.org/urn:doi:10.3390/ijms242015436
url https://ddd.uab.cat/record/300101
https://dx.doi.org/urn:doi:10.3390/ijms242015436
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.relation.none.fl_str_mv Instituto de Salud Carlos III https://doi.org/10.13039/501100004587 DTS18/00101
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
https://creativecommons.org/licenses/by/4.0/
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
https://creativecommons.org/licenses/by/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv reponame:Dipòsit Digital de Documents de la UAB
instname:Universitat Autònoma de Barcelona
instname_str Universitat Autònoma de Barcelona
reponame_str Dipòsit Digital de Documents de la UAB
collection Dipòsit Digital de Documents de la UAB
repository.name.fl_str_mv
repository.mail.fl_str_mv
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