Data normalization of plasma miRNA profiling from patients with COVID-19

• Published in: Scientific reports 2024-11, Vol.14 (1), p.26791-12, Article 26791
• Main Authors: Siguemoto, Julia Tiemi, Motta Neri, Carolini, de Godoy Torso, Nadine, de Souza Nicoletti, Aline, Berlofa Visacri, Marília, Regina da Silva Correa da Ronda, Carla, Perroud, Mauricio Wesley, Oliveira Reis, Leonardo, dos Santos, Luiz Augusto, Durán, Nelson, Fávaro, Wagner José, de Carvalho Pincinato, Eder, Moriel, Patricia
• Format: Article
• Language: English
• Subjects: 631/1647; 631/1647/2163; 631/1647/2210; 631/337; 631/337/176; 631/337/384; Adult; Aged; Case-Control Studies; Coronaviruses; COVID-19; COVID-19 - blood; COVID-19 - genetics; COVID-19 - virology; Endogenous normalizer; Female; Gene Expression Profiling - methods; Humanities and Social Sciences; Humans; Male; MicroRNA; MicroRNAs; MicroRNAs - blood; MicroRNAs - genetics; Middle Aged; miRNA; multidisciplinary; Reference gene; RNA-Seq - methods; SARS-CoV-2 - genetics; Science; Science (multidisciplinary)
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-024-75740-3

When using the reverse-transcription quantitative polymerase chain reaction (RT-qPCR) technique for quantitative assessment of microRNA (miRNA) expression, normalizing data using a stable endogenous gene is essential; however, no universally adequate reference gene exists. Therefore, in this study, we aimed to determine, via the RNA-Seq technique, the most adequate endogenous normalizer for the expression assessment of plasma miRNAs in patients with coronavirus disease 2019 (COVID-19). Two massive sequencing procedures were performed (a) to identify differentially expressed miRNAs between patients with COVID-19 and healthy volunteers ( n =  12), and (b) to identify differentially expressed miRNAs between patients with severe COVID-19 and those with mild COVID-19 ( n =  8). The endogenous normalizer candidates were selected according to the following criteria: (1) the miRNA must have a fold regulation = 1; (2) the miRNA must have a p -value > 0.990; and (3) the miRNAs that were discovered the longest ago should be selected. Four miRNAs (hsa-miR-34a-3p, hsa-miR-194-3p, hsa-miR-17-3p, and hsa-miR-205-3p) met all criteria and were selected for validation by RT-qPCR in a cohort of 125 patients. Of these, only hsa-miR-205-3p was eligible endogenous normalizers in the context of COVID-19 because their expression was stable between the compared groups.