RESUMO
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.
Assuntos
MicroRNAs , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA-Seq , Análise de Sequência de RNA , Sequência de Bases , Isoformas de Proteínas/metabolismoRESUMO
Este trabalho aborda as distintas estratégias construídas em rede por meio de cooperação horizontal adotadas pelo município de Diadema, para subsidiar as equipes mediante a complexidade que é ofertar a longitudinalidade do cuidado aos pacientes com suspeita /diagnóstico de sífilis. Dessa forma, o principal objetivo é apoiar, aperfeiçoar e padronizar a assistência ofertada pelas equipes da Atenção Básica com arranjos de baixo custo nas ações de prevenção, detecção, tratamento, monitoramento e combate à sífilis adquirida, em gestante e congênita, de acordo com as diretrizes do Ministério da Saúde. O progresso das estratégias utilizadas demonstrou impactos positivos a respeito do rastreamento e tratamento da sífilis no município, em médio prazo e custo relativamente baixo.
