3'IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR : Impact on Small RNA-Seq Profiling Validation
Ferre-Giraldo, Adriana (Institut d'Investigació Biomèdica de Bellvitge)
Santiago, Lucía (Institut d'Investigació Biomèdica de Bellvitge)
Sánchez-Herrero, Jose Francisco (Institut Germans Trias i Pujol. Hospital Universitari Germans Trias i Pujol)
López-Rodrigo, Olga (Fundació Puigvert)
Sánchez-Curbelo, Josvany (Institut d'Investigació Biomèdica Sant Pau)
Sumoy, Lauro (Institut Germans Trias i Pujol. Hospital Universitari Germans Trias i Pujol)
Bassas, Lluís (Institut d'Investigació Biomèdica Sant Pau)
Larriba, Sara (Institut d'Investigació Biomèdica de Bellvitge)
Universitat Autònoma de Barcelona

Date:	2023
Abstract:	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.
Grants:	Instituto de Salud Carlos III DTS18/00101
Rights:	Aquest 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.
Language:	Anglès
Document:	Article ; recerca ; Versió publicada
Subject:	IsomiRs ; MiRNA isoforms ; Poly(A)-RT-qPCR ; Selective amplification ; Small RNAseq validation strategy
Published in:	International journal of molecular sciences, Vol. 24 Núm. 20 (october 2023) , p. 15436, ISSN 1422-0067

DOI: 10.3390/ijms242015436
PMID: 37895116

14 p, 1.0 MB

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Research literature > UAB research groups literature > Research Centres and Groups (research output) > Health sciences and biosciences > Institut d'Investigació en Ciencies de la Salut Germans Trias i Pujol (IGTP)
Research literature > UAB research groups literature > Research Centres and Groups (research output) > Health sciences and biosciences > Institut de Recerca Sant Pau
Articles > Research articles
Articles > Published articles