Gynecology and Obstetrics Department, Ribeirao Preto Medical School, University of Sao Paulo, Bandeirantes Avenue, 3900, 8th Floor of the Clinic Hospital, Universitary Campus, Ribeirao Preto, SP, 14049-900, Brazil.
Multiuser Laboratory, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP, Brazil.
Sci Rep. 2021 Mar 8;11(1):5422. doi: 10.1038/s41598-021-84884-5.
It has been suggested that menstrual blood-derived mesenchymal stem/stromal cells (MenMSCs) are associated with the etiopathogenesis of endometriosis and considerable effort has been invested in searching for target genes and deciphering associated molecular pathways. However, reference gene stability for proper reproducible normalization in the analyses of the expression data validation is still unexplored in this experimental context. Therefore, in this exploratory study, we used stringent case and control selection criteria and collected menstrual blood from women with a laparoscopic diagnosis of advanced endometriosis and from fertile women without endometriosis. We tested for the first time the stability of 32 candidate reference genes to achieve increased accuracy and reliable results in the quantification of gene expression and direct future experiments using reverse transcription-quantitative PCR (RT-qPCR) in MenMSCs for endometriosis studies. Using the RefFinder web tool, we recommend the EIF2B1 and POP4 reference genes for the normalization of RT-qPCR data in study designs similar to ours. Furthermore, we suggest avoiding the commonly used GAPDH and ACTB reference genes as they are unstable. This high-visibility study is capable of directing different experimental designs as MenMSCs are derived from a minimally invasive tissue source with multifunctional roles in regenerative medicine.
有人提出，月经血源性间充质干细胞（MenMSCs）与子宫内膜异位症的病因发病机制有关，人们已经投入了大量的精力来寻找靶基因并阐明相关的分子途径。然而，在这种实验背景下，对于适当的可重复表达数据验证分析中的参考基因稳定性仍然没有得到探索。因此，在这项探索性研究中，我们使用严格的病例和对照选择标准，收集了腹腔镜诊断为晚期子宫内膜异位症的女性和无子宫内膜异位症的生育期女性的月经血。我们首次测试了 32 个候选参考基因的稳定性，以提高在 MenMSCs 中定量基因表达的准确性和可靠性，并直接为未来的逆转录定量 PCR（RT-qPCR）实验提供参考，用于子宫内膜异位症的研究。使用 RefFinder 网络工具，我们建议在与我们的研究设计类似的研究中，使用 EIF2B1 和 POP4 参考基因进行 RT-qPCR 数据的归一化。此外，我们建议避免使用常用的 GAPDH 和 ACTB 参考基因，因为它们不稳定。这项高可见度的研究能够指导不同的实验设计，因为 MenMSCs 来源于一种微创的组织来源，在再生医学中具有多功能作用。
