Cargando…
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...
Autores principales: | , , , , , , , |
---|---|
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 |
_version_ | 1785127482774519808 |
---|---|
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. |
format | Online Article Text |
id | pubmed-10607168 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT ferreadriana 3isomirspeciescompositionaffectsreliablequantificationofmirnaisomirvariantsbypolyartqpcrimpactonsmallrnaseqprofilingvalidation AT santiagolucia 3isomirspeciescompositionaffectsreliablequantificationofmirnaisomirvariantsbypolyartqpcrimpactonsmallrnaseqprofilingvalidation AT sanchezherrerojosefrancisco 3isomirspeciescompositionaffectsreliablequantificationofmirnaisomirvariantsbypolyartqpcrimpactonsmallrnaseqprofilingvalidation AT lopezrodrigoolga 3isomirspeciescompositionaffectsreliablequantificationofmirnaisomirvariantsbypolyartqpcrimpactonsmallrnaseqprofilingvalidation AT sanchezcurbelojosvany 3isomirspeciescompositionaffectsreliablequantificationofmirnaisomirvariantsbypolyartqpcrimpactonsmallrnaseqprofilingvalidation AT sumoylauro 3isomirspeciescompositionaffectsreliablequantificationofmirnaisomirvariantsbypolyartqpcrimpactonsmallrnaseqprofilingvalidation AT bassaslluis 3isomirspeciescompositionaffectsreliablequantificationofmirnaisomirvariantsbypolyartqpcrimpactonsmallrnaseqprofilingvalidation AT larribasara 3isomirspeciescompositionaffectsreliablequantificationofmirnaisomirvariantsbypolyartqpcrimpactonsmallrnaseqprofilingvalidation |