PDF(Pubmed)
Abstract：
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.
摘要：
在大多数研究中，选择合适的内参基因或内部对照来标准化RT-qPCR数据对于通过RT-qPCR获得的基因表达数据的实验间重复性是强制性的。包括子宫内膜异位症.特别是对于miRNA，由于其物理化学和生物学特性，选择参考基因具有挑战性。此外，月经逆行理论，经血中的间充质干细胞（MenSCs），以及通过miRNAs的转录后调控过程的变化在科学界作为子宫内膜异位症的重要参与者而得到了重视。因此,我们最初探索了10种miRNA表达在涉及健康女性和子宫内膜异位症患者MenSCs二维培养的条件下作为内部对照候选物的稳定性。这里,我们应用了多种算法(geNorm，NormFinder,最佳管理员,和deltaCt）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计算的成对变异将三个miRNA鉴定为用于精确归一化的足够数量的参考基因。MiR-191-5p,miR-24-3p,和miR-103a-3p是合适基因表达正常化的最佳组合。这项研究将有利于类似的研究，但对于使用MenSCs的再生医学和诊所也很有吸引力，miRNA表达，和RT-qPCR。
