Ferré, AdrianaSantiago, LucíaSánchez Herrero, José FranciscoLópez Rodrigo, OlgaSánchez Curbelo, JosvanySumoy, LauroBassas, LluísLarriba, Sara2024-03-262024-03-262023-10-211422-0067https://hdl.handle.net/2445/209210Small 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.14 p.application/pdfengcc by (c) Ferré, Adriana et al., 2023http://creativecommons.org/licenses/by/3.0/es/Micro RNAsCàncerMicroRNAsCancerinfo:eu-repo/semantics/article2023-11-16info:eu-repo/semantics/openAccess37895116