Selection of reference miRNAs for RT-qPCR assays in endometriosis menstrual blood-derived mesenchymal stem cells

• Published in: PloS one 2024-07, Vol.19 (7), p.e0306657
• Main Authors: Hacimoto, Sabrina Yukari Santos, Cressoni, Ana Clara Lagazzi, Silva, Lilian Eslaine Costa Mendes da, Padovan, Cristiana Carolina, Ferriani, Rui Alberto, Rosa-E-Silva, Júlio César, Meola, Juliana
• Format: Article
• Language: English
• Subjects: Adult; Algorithms; Biology and life sciences; Blood; Cell culture; Chromosome 3; Chromosome 5; Computer and Information Sciences; Control stability; Dimensional stability; Disease; Endometriosis; Endometriosis - genetics; Engineering and Technology; Ethics; Ethylenediaminetetraacetic acid; Female; Gene expression; Gene Expression Profiling - methods; Genes; Humans; Instrument industry; Medical research; Medicine and Health Sciences; Menstruation; Menstruation - genetics; Mesenchymal stem cells; Mesenchymal Stem Cells - metabolism; MicroRNA; MicroRNAs; MicroRNAs - genetics; miRNA; Molecular biology; Physical Sciences; Post-transcription; Real-Time Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - standards; Reference Standards; Regenerative medicine; Reproducibility of Results; Research and Analysis Methods; Stem cells; Womens health
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0306657

Choosing appropriate reference genes or internal controls to normalize RT-qPCR data is mandatory for the interexperimental reproducibility of gene expression data obtained by RT-qPCR in most studies, including those on endometriosis. Particularly for miRNAs, the choice for reference genes is challenging because of their physicochemical and biological characteristics. Moreover, the retrograde menstruation theory, mesenchymal stem cells in menstrual blood (MenSCs), and changes in post-transcriptional regulatory processes through miRNAs have gained prominence in the scientific community as important players in endometriosis. Therefore, we originally explored the stability of 10 miRNAs expressions as internal control candidates in conditions involving the two-dimensional culture of MenSCs from healthy women and patients with endometriosis. Here, we applied multiple algorithms (geNorm, NormFinder, Bestkeeper, and delta Ct) to screen reference genes and assessed the comprehensive stability classification of miRNAs using RefFinder. Pairwise variation calculated using geNorm identified three miRNAs as a sufficient number of reference genes for accurate normalization. MiR-191-5p, miR-24-3p, and miR-103a-3p were the best combination for suitable gene expression normalization. This study will benefit similar research, but is also attractive for regenerative medicine and clinics that use MenSCs, miRNA expression, and RT-qPCR.