gender: female disease severity: minimal endometriosis cell treatment: peritoneal fluid cell line treated: telomerase-immortalized human endometrial stromal cells
Treatment protocol
Telomerase – immortalized human endometrial stromal cells (T-HESC; ATCC CRL-4003) were maintained in Dulbecco’s Modified Eagle’s medium (DMEM) with charcoal stripped 10% FBS, insulin, selenious acid, transferrin, BSA, and linoleic acid (ITS+Premix), puromycin and pen/strep. Cells were plated for experiments at 80% confluence followed by starvation for 6 hours. Peritoneal fluid was aspirated from human subjects without and with endometriosis who were undergoing pelvic surgery. This study was approved by the University of South Dakota Institutional Review Board and informed consent was obtained from all subjects. The stage of endometriosis of each patient was classified using the American Society for Reproductive Medicine paradigm (minimal, mild, moderate or severe). The fluid was centrifuged and filtered through a 0.22 micron filter to remove cells and debris. The filtered peritoneal fluid was combined with T-HESC growth medium (DMEM, 4:1) with growth supplements (ITS) and antibiotics (penicillin/streptomycin). The peritoneal fluid/growth medium was added to T-HESC cells that had been starved for 6 hours, and incubation was continued for 48 hours. After 48 hours of treatment, T-HESC were harvested for RNA isolation.
Extracted molecule
total RNA
Extraction protocol
Treated cultured T-HESC cells were lysed in 1 ml TRI reagent. Bromochloropropane and sodium acetate were added, and the samples were centrifuged to separate the phases. The RNA-containing layer was removed and the RNA purified on an RNeasy extraction column (Qiagen). The sample was treated with an on-column DNase treatment (RNase-free DNase, Qiagen) and the purified RNA was eluted. The purity and quantity of RNA were evaluated by an Agilent Bioanalyzer using the RNA 6000 Nanoassay LabChip.
Label
streptavidin Alexa Fluor 647
Label protocol
Labeled cRNA was prepared using the MessageAmp II-Biotin enhanced kit (Ambion). 1.0 microgram of total RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C. (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.) The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C. The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C. The double-stranded cDNA was purified on columns. The double-stranded cDNA was mixed with T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA. The cRNA synthesis reaction was terminated after 14h at 37C by purifying the cRNA. The concentration of cRNA was determined by spectrophotometry. Streptavidin Alexa fluor 647 was incubated with the slides after hybridization to complete the labeling reaction. One sample was hybridized with each slide so no dye swaps were necessary.
Hybridization protocol
10 micrograms of cRNA was mixed with fragmentation buffer and heated to 94C for 20 min. The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the microarray slides, and hybridized for 18 hours at 37C. The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature. The Alexa fluor was prepared in TNB blocking buffer (0.1M Tris-HCl, 0.15M NaCL, 0.5% NEN Blocking Reagent-PerkinElmer) The slides were then washed 4 times for 5 min each in 1x TNT and once in 0.05% Tween 20 for 5 sec each. The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner.
Scan protocol
The slides were scanned in an Axon GenePix 4000B scanner using GenePix Pro 4.1 to align and acquire the microarray image. CodeLink Expression Analysis 4.1.0.4163 was used to apply the background correction.
Data processing
Data in the matrix table were prepared by global median normalization applied by CodeLink Expression Analysis 4.1.0.4163.
