Tissue Processing and Fluorescence Activated Cell Sorting (FACS) of Endometrial Cell Populations: Tissue biopsies were divided into two fresh tissue samples processed separately for fluorescence activated cell sorting (FACS) and for histological examination in formalin-fixed, paraffin embedded tissue. Tissue processing for viable cell isolation and FACS analysis were performed as previously described (15). Briefly, enzymatically dissociated endometrial cells were incubated in blocking buffer (PBS with 40% human serum [HS] and 1% bovine serum albumin [BSA]) for 30min then labeled with the following fluorochrome-conjugated antibodies (BD Biosciences, San Jose, CA) in PBS containing 10% HS and 1% BSA: cluster of differentiation 45 (CD45, PE-Cy7 anti-CD45) at 1:20 dilution to label contaminating leukocytes for their removal; epithelial cell adhesion molecule (EPCAM, allophycocyanin anti-EPCAM) at 1:20 dilution to label eEP; cluster of differentiation 146 (CD146 or melanoma cell adhesion molecule [MCAM], CD146, fluorescein isothiocyanate anti-MCAM) at 1:5 dilution to label eEN/perivascular cells; beta-type platelet-derived growth factor receptor (PDGFRB, phycoerythrin (PE) anti-PDGFRB) at 1:5 dilution to label eSF. eMSCs were sorted using double labeling for CD146 and PDGFRB antibodies respectively, both at 1:5 dilutions. The cell suspension was sorted using a FACS Aria II with FACS Diva software (BD Biosciences). The FACS-sorted cell pellets were stored at -80C until RNA extraction. FACS analyzed sorted cell populations were subject to RNA isolation and purification, with DNAse treatment, using Pico Pure RNA Isolation Kit
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated form FACS-sorted cell populations and purified using the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA) following manufacturer´s instructions. An additional DNase treatment was performed using the RNase-Free DNase Set (Qiagen, Valencia, CA).
Label
Biotin
Label protocol
Reverse transcription and amplification of isolated RNA into cDNA was performed using NuGEN WT-Ovation Exon FFPE System V2 (NuGen, San Carlos, CA). The integrity of resultant cDNA was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies), and individual samples meeting yield and quality standards were further processed and hybridized to Affymetrix Human Gene 1.0 ST arrays (Affymetrix, Cleveland, OH), probing 36,079 genes.
Hybridization protocol
Microarrays were hybridized, washed, stained, and scanned according to the protocol described in the WT sense target labeling assay manual from Affymetrix (version 4; FS450_0007).
Scan protocol
Microarrays were hybridized, washed, stained, and scanned according to the protocol described in WT Sense Target Labeling Assay Manual from Affymetrix (Version 4; FS450_0007) at the UCSF Gladstone Genomics Core Facility.
Data processing
GeneSpringGX 11, Microarray Technology: Affymetrix.ExonExprChip.HuGene-1_0-st-v1, Summarization Algorithm: ExonRMA16, Normalization: Quantile, Baseline Transformation: median of all samples Summarization Algorithm: RMA16. Transcript Level: Core. Baseline to median of all samples
Mesenchymal Stem/Progenitors and Other Endometrial Cell Types from Women with Polycystic Ovary Syndrome (PCOS) Display Inflammatory and Oncogenic Potential
