Duplicate cultures of hESF in LSM were treated with E2P4 (10 nM E2 + 1 μM P4), with 10 nM E2 alone (E2), or with vehicle (vehicle control), and cells and CM were collected at time (t) of 0 h, 6 h (early response), 48 h (intermediate response), and 14 days (late response). CM was changed every 2 days.
Growth protocol
Endometrial tissue was digested with collagenase, and hESFs were isolated and plated with Dulbecco's Modified Eagle medium (DMEM)/molecular cell developmental biology 105 (MCDB-105) medium with 10% charcoal-stripped fetal bovine serum (FBS), insulin (5 mg/ml), gentamicin, penicillin, and streptomycin. All cells used were at Passage 2. Subsequently, hESFs were plated in 6-cm plastic dishes, and after they reached confluency, medium was changed to a low-serum medium (LSM; i.e., DMEM/MCDB-105 medium containing ascorbic acid, transferrin, and gentamicin with 2% FBS) for 24 h prior to treatment.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from individual plates and purified using an RNeasy mini-kit (Qiagen, Valencia, CA) following the manufacturer's protocol. Samples were stored in RNase-free H2O, purity was analyzed by the A260:A280 nm ratio, and RNA quality and integrity were assessed by using a Bioanalyzer 2100 unit (Agilent Technologies, Santa Clara, CA). All samples had high-quality RNA (RNA integrity number [RIN] = 9.7–10).
Label
Biotin
Label protocol
For each sample, 100 ng of total RNA was reverse-transcribed to cDNA by using 500 ng of T7-(N6) primer and SuperScript II. A second strand of DNA was generated using DNA polymerase, followed by overnight in vitro transcription to generate cRNA. After samples were processed through cRNA clean-up spin columns (Affymetrix), 10 μg of cRNA was reverse-transcribed using random primers and SuperScript II. Mixtures were digested with RNase H, and cDNA was purified by cDNA clean-up spin columns (Affymetrix). Finally, 5.5 μg of sense cDNA was fragmented and labeled using a GeneChip WT-terminal labeling kit. The quality of the cDNA and the fragmented cDNA was assessed with an Agilent Bioanalyzer.
Hybridization protocol
Individual samples were hybridized to Human Gene 1.0 ST arrays (Affymetrix) containing 19,492 genes.
Scan protocol
Data were scanned according to the protocol described in the WT sense target-labeling assay manual from Affymetrix (version 4; product code FS450_0007).
Description
61 LUSY61
Data processing
The intensity values of different probe sets (genes) in the GeneChip Operating Software (Affymetrix) were imported into GeneSpring GX version 10.0 software (Agilent Technologies, Santa Clara, CA) and processed using the robust multiarray analysis (RMA) algorithm for background adjustment, normalization, and log2 transformation of perfect-match values. The data at each time point were normalized to the corresponding sample at t = 0 h. Then, the normalization was conducted within each time-group: data from E2P4-treated samples were normalized to those from E2-treated samples, which were normalized to the vehicle control, with the resulting data thus reflecting the specific temporal response to P4. We performed three major comparisons of the response to P4: within the nonendometriosis group, within the endometriosis group, and between hESFendo and hESFnonendo. The resulting gene lists generated included only genes showing >1.5-fold change in expression and a P value of <0.05 by using a two-way ANOVA parametric test and Benjamini-Hochberg multiple testing correction for false discovery rate.
