He study.RNA isolation and quality controlMethods The study protocol wasHe study.RNA isolation and quality controlMethods

He study.RNA isolation and quality controlMethods The study protocol was
He study.RNA isolation and quality controlMethods The study protocol was approved by the Local Ethical Committee of Medical University of Bialystok, Poland, and signed informed consent was obtained from each patient. A total of 46 patients were recruited in this study. Endometrial samples were initially obtained from 51 regularly menstruating women, aged 20?5 years, undergoing diagnostic or surgical laparoscopic surgery for non-malignant ovarian lesions. After total RNA isolation and quality control (see RNA isolation and quality control), we selected 46 samples for further molecular analysis. Patients with endometriosis (Group I, n = 21) stages III to IV were diagnosed through laparoscopic findings according to the revised American Fertility Society classification of endometriosis [14], and each case was confirmed through histopathology. As a control (Group II, n = 25), we used endometrial tissue from 16 women who underwent surgery as part of an infertility work-up and 9 women who underwent surgery to remove simple ovarian cysts; all controls were without any visible endometriosis during laparoscopy. Patients with autoimmune disease, pelvic inflammatory disease, adenomyosis, dysfunctional uterine bleeding and those taking non-steroidal anti-inflammatory drugs, GnRH agonists and steroids for the past 3 months were excluded. Endometrial samples were collectedA piece of each collected tissue was divided into two halves and placed separately in buffered formaline for histopathological studies and RNAlater solution (Sigma Aldrich, Poland) for PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28381880 molecular analysis. The latter was stored for 24 hours at +4 , and subsequently the tissues were transferred and stored at -80 . Total RNA was extracted using the mirVanaTM miRNA Isolation Kit (Ambion, Life Technologies, Poland). The RNA quality was assessed using an Agilent Bioanalyzer 2100 and Agilent RNA 6000 Nano kit (Agilent Technologies, Perlan, Poland), and the samples selected for further analysis showed minimum degradation, with an RNA integrity number (RIN) above 9 for all samples. The five excluded samples had RINs between 4 and 6. The RNA concentrations were measured using a NanoDrop 2000c (Thermo Scientific, Biotech, Poland). We also used small RNA microfluidic chips (Agilent Small RNA kit, Agilent Technologies, Perlan, Poland), and only the visual evaluation of an apparent band on the capillary gel of electropherograms was performed, as no algorithm PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27797473 has yet been created for this purpose.TaqMan MicroRNA Array CardsThe TaqMan low-density Arrays A and B (TaqMan?Array Human MicroRNA A v2.1 + B v2.0 Cards Set, Applied Biosystems, Life Technologies, Poland), which I-CBP112MedChemExpress I-CBP112 facilitate the comprehensive coverage of Sanger miRBase v14, were configured in a 384-well format microRNA assays spotted onto a microfluidic card, and these analyses were performed on 10 samples of the eutopic endometrium of patients with ovarian endometriosis and 10 controls. The Micro Fluidic Card contains eight sample-loading ports, each connected by a micro channel to 48 miniature reaction chambers for a total of one sample per card. The details are available at: http://tools.invitrogen.com/content/ sfs/manuals/cms_054742.pdf. Single-stranded cDNA was synthesised from 500 ng of total RNA using the TaqMan?MicroRNA Reverse Transcription Kit (Applied Biosystems, Life Technologies, Poland) and highly multiplexed RNA-specific stemlooped Megaplex RT Primers, Human Pool A v2.1 and Human Pool B v2.0 (Applied Biosystems, Life Technologies.