Endometrial tissue biopsies were obtained from 14 reproductive age women. Six subjects had endometriosis (n=1 minimal, n=2 mild, n=1 moderate-severe, n=2 severe), on visualization of lesions during laparoscopy and their histologic evaluation (Table 1). Staging of endometriosis was according to the revised American Fertility Society classification system (19). Participating subjects with endometriosis were 22-46 years old (mean 33.5+/-3.0), not pregnant, and did not use hormonal medications within 3 months before surgery. Controls were eight cycling subjects (37-49 years old, mean 44.4+/-.7) undergoing endometrial biopsy or hysterectomy for benign reasons such as fibroids, pelvic organ prolapse, pelvic pain or endometrial polyps. Controls had regular menstrual cycles (25-35 days), were documented not to be pregnant, had no history of endometriosis and no evidence of endometriosis at laparoscopy and had not been on hormonal therapies for at least 3 months before tissue sampling.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from hESF from subjects without (n=4) and with (n=4) endometriosis and purified using Qiagen RNeasy Plus Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Samples were stored in RNase-free H2O, quantified by spectroscopy, and purity was analyzed using the 260/280 absorbance ratio. RNA quality and integrity were assessed using Agilent Bioanalyser 2100 (Agilent Technologies, Santa Clara, CA, USA) with all samples demonstrating high quality (RIN=9.7-10).
Label
biotin
Label protocol
Hybridization was performed using Affymetrix Human Gene 1.0 ST arrays (Affymetrix, Inc, Santa Clara, CA). Briefly, for each sample 100 ng of total RNA were reverse transcribed to cDNA using 500 ng of T7-(N6) primers and SuperScript II. A second strand of DNA was generated using DNA Polymerase, followed by overnight in vitro transcription to generate cRNA. After processing through cRNA cleanup spin columns, 10 μg of cRNA was reverse transcribed using random primers and SuperScript II. Mixtures were digested with RNase H, and the cDNA purified by the cDNA clean-up spin columns. Finally, 5.5 μg of sense cDNA was fragmented and labeled using the geneChip WT terminal labeling kit. Quality of cDNA and fragmented cDNA was assessed using an Agilent bioanalyzer.
Hybridization 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).
Scan protocol
According to manufacturer's instruction.
Description
No additional information
Data processing
To minimize technical (non-biological) variability among arrays, densitometry values between arrays were normalized using the RMA function and further transformed to the logarithmic scale (log2). Probes with a corresponding GenBank accession ID were selected for functional analysis. Statistically significant differences between groups were determined using SAM and RankProd methods using the Bioconductor (http://www.bioconductor.org/) packages Siggene and RankProd, respectively, both run under R software (http://www.r-project.org/).
