Department of Gynecology and Obstetrics of Ribeirao Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.
National Institute of Hormones and Women's Health (Hormona), CNPq, Porto Alegre, Rio Grande do Sul, Brazil.
PLoS One. 2024 Jul 30;19(7):e0306657. doi: 10.1371/journal.pone.0306657. eCollection 2024.
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.
选择合适的参考基因或内参来标准化 RT-qPCR 数据，对于大多数研究中通过 RT-qPCR 获得的基因表达数据的实验间可重复性至关重要，包括子宫内膜异位症的研究。特别是对于 miRNA，由于其理化和生物学特性，选择参考基因具有挑战性。此外，逆行月经理论、月经血中的间充质干细胞（MenSCs）以及 miRNA 在后转录调控过程中的变化，作为子宫内膜异位症的重要参与者，在科学界引起了关注。因此，我们最初探索了在二维培养健康女性和子宫内膜异位症患者的 MenSCs 条件下，10 种 miRNA 表达作为内参候选物的稳定性。在这里，我们应用了多种算法（geNorm、NormFinder、Bestkeeper 和 delta Ct）来筛选参考基因，并使用 RefFinder 评估 miRNA 的综合稳定性分类。使用 geNorm 计算的成对变化确定了三个 miRNA 作为准确归一化的足够数量的参考基因。miR-191-5p、miR-24-3p 和 miR-103a-3p 是适合基因表达归一化的最佳组合。这项研究将有益于类似的研究，但也吸引了再生医学和使用 MenSCs、miRNA 表达和 RT-qPCR 的临床应用。
