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...

Descripción completa

Detalles Bibliográficos
Autores: Ferre, A, Santiago, L, Sánchez-Herrero, JF, López-Rodrigo, O, Sánchez-Curbelo, J, Sumoy, L, Bassas, L, Larriba, S
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2023
País:España
Institución:Institut d’Investigació Biomèdica Sant Pau (IIB Sant Pau)
Repositorio:r-IIB SANT PAU. Repositorio Institucional de Producción Científica del Instituto de Investigación Biomédica Sant Pau
OAI Identifier:oai:iibsantpau.fundanetsuite.com:p17311
Acceso en línea:https://iibsantpau.fundanetsuite.com/Publicaciones/ProdCientif/PublicacionFrw.aspx?id=17311
Access Level:acceso abierto
Palabra clave:miRNA isoforms
isomiRs
poly(A)-RT-qPCR
selective amplification
small RNAseq validation strategy
id ES_3e9df78a95aea284cd0d8bbe805d6d3d
oai_identifier_str oai:iibsantpau.fundanetsuite.com:p17311
network_acronym_str ES
network_name_str España
repository_id_str
spelling 3'IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR: Impact on Small RNA-Seq Profiling ValidationFerre, ASantiago, LSánchez-Herrero, JFLópez-Rodrigo, OSánchez-Curbelo, JSumoy, LBassas, LLarriba, SmiRNA isoformsisomiRspoly(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.MDPI2023info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttps://iibsantpau.fundanetsuite.com/Publicaciones/ProdCientif/PublicacionFrw.aspx?id=17311INTERNATIONAL JOURNAL OF MOLECULAR SCIENCESISSN: 16616596ISSNe: 14220067reponame:r-IIB SANT PAU. Repositorio Institucional de Producción Científica del Instituto de Investigación Biomédica Sant Pauinstname:Institut d’Investigació Biomèdica Sant Pau (IIB Sant Pau)Inglésinfo:eu-repo/semantics/openAccessoai:iibsantpau.fundanetsuite.com:p173112026-06-14T12:41:47Z
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
Ferre, A
miRNA isoforms
isomiRs
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: 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 Ferre, A
Santiago, L
Sánchez-Herrero, JF
López-Rodrigo, O
Sánchez-Curbelo, J
Sumoy, L
Bassas, L
Larriba, S
author Ferre, A
author_facet Ferre, A
Santiago, L
Sánchez-Herrero, JF
López-Rodrigo, O
Sánchez-Curbelo, J
Sumoy, L
Bassas, L
Larriba, S
author_role author
author2 Santiago, L
Sánchez-Herrero, JF
López-Rodrigo, O
Sánchez-Curbelo, J
Sumoy, L
Bassas, L
Larriba, S
author2_role author
author
author
author
author
author
author
dc.subject.none.fl_str_mv miRNA isoforms
isomiRs
poly(A)-RT-qPCR
selective amplification
small RNAseq validation strategy
topic miRNA isoforms
isomiRs
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 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://iibsantpau.fundanetsuite.com/Publicaciones/ProdCientif/PublicacionFrw.aspx?id=17311
url https://iibsantpau.fundanetsuite.com/Publicaciones/ProdCientif/PublicacionFrw.aspx?id=17311
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv MDPI
publisher.none.fl_str_mv MDPI
dc.source.none.fl_str_mv INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
ISSN: 16616596
ISSNe: 14220067
reponame:r-IIB SANT PAU. Repositorio Institucional de Producción Científica del Instituto de Investigación Biomédica Sant Pau
instname:Institut d’Investigació Biomèdica Sant Pau (IIB Sant Pau)
instname_str Institut d’Investigació Biomèdica Sant Pau (IIB Sant Pau)
reponame_str r-IIB SANT PAU. Repositorio Institucional de Producción Científica del Instituto de Investigación Biomédica Sant Pau
collection r-IIB SANT PAU. Repositorio Institucional de Producción Científica del Instituto de Investigación Biomédica Sant Pau
repository.name.fl_str_mv
repository.mail.fl_str_mv
_version_ 1869406558391107584
score 15,812429

Similares en LA Referencia