3'IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR: Impact on Small RNA-Seq Profiling Validation
Por:
Ferre, A, Santiago, L, Sánchez-Herrero, JF, López-Rodrigo, O, Sánchez-Curbelo, J, Sumoy, L, Bassas, L, Larriba, S
Publicada:
1 oct 2023
Resumen:
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.
Filiaciones:
Ferre, A:
Bellvitge Biomed Res Inst IDIBELL, Human Mol Genet Grp, Lhospitalet De Llobregat 08908, Spain
Santiago, L:
Bellvitge Biomed Res Inst IDIBELL, Human Mol Genet Grp, Lhospitalet De Llobregat 08908, Spain
Sánchez-Herrero, JF:
Germans Trias i Pujol Res Inst IGTP, High Content Genom & Bioinformat HCGB, Badalona 08916, Spain
López-Rodrigo, O:
Androl Serv Puigvert Fdn, Lab Androl & Sperm Bank, Barcelona 08025, Spain
Sánchez-Curbelo, J:
Androl Serv Puigvert Fdn, Lab Androl & Sperm Bank, Barcelona 08025, Spain
Sumoy, L:
Germans Trias i Pujol Res Inst IGTP, High Content Genom & Bioinformat HCGB, Badalona 08916, Spain
Bassas, L:
Androl Serv Puigvert Fdn, Lab Androl & Sperm Bank, Barcelona 08025, Spain
Larriba, S:
Bellvitge Biomed Res Inst IDIBELL, Human Mol Genet Grp, Lhospitalet De Llobregat 08908, Spain
Green Published, gold
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