|
| Status |
Public on Jan 01, 2016 |
| Title |
decidual cells, vehicle, biological rep2 |
| Sample type |
RNA |
| |
|
| Source name |
decidual cells treated with vehicle
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: decidua
cell type: fibroblast
agent: vehicle
time: 6 h
|
| Treatment protocol |
The cultures were treated with PBS containing 0.1% bovine serum albumin (vehicle control) or with 1 ng/ml of IL-1β (R&D Systems) for 6 h.
|
| Growth protocol |
After written Institutional Review Board approval and informed consent, fibroblasts were prepared from the decidua parietalis of reflected fetal membranes obtained from uncomplicated pregnancies after elective repeat cesarean deliveries. All specimens were obtained prior to the onset of labor. The decidua was scraped from the maternal surface of the chorion, minced, and incubated with 25 mg/ml of collagenase (200 U/mg) (Worthington Biochemical) and 6.25 U/ml of DNase (Sigma-Aldrich). Cell clusters in the final digestate were further disrupted by passage through a 23-gauge needle. The cells were enriched by centrifugation through discontinuous Percoll gradients, and then cultivated over three passages until >99% free of CD45+ leukocytes in basal medium, a phenol red-free 1:1 vol/vol mix of Dulbecco's Modified Eagle Medium (DMEM)/Ham’s F-12 (Life Technologies) with 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B supplemented with 10% charcoal stripped calf serum (SCS). Passaged, confluent, leukocyte-free decidual cells were primed for 13 days in basal medium supplemented with SCS containing 10-8 M 17β-estradiol (E2; Sigma-Aldrich) and 10-7 M medroxyprogesterone acetate (MPA; Sigma-Aldrich). The cultures were then washed twice with PBS and switched to a defined medium consisting of basal medium (without the addition of SCS) plus ITS+ Premix (BD Biosciences), 5 μM FeSO4, 0.5 μM ZnSO4, 1 nM CuSO4 (Sigma-Aldrich), 50 μg/ml ascorbic acid (Sigma-Aldrich) and 50 ng/ml epidermal growth factor (BD Biosciences) with E2 and MPA overnight prior to experiments. Cells from three different donors were used for the studies.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with TRIzol (Invitrogen) and after the addition of chloroform and centrifugation, the aqueous phase was applied to a miRNeasy spin column (Qiagen) and processed according to the manufacturer’s protocol. This included on-column DNase digestion using the RNase-Free DNase kit (Qiagen) to remove contaminating genomic DNA.
|
| Label |
biotin
|
| Label protocol |
250 nanograms of total RNA from each sample were used to prepare biotinylated fragmented cRNA according to the GeneChip Expression. Analysis Technical Manual (Rev. 5, Affymetrix Inc., Santa Clara, CA).
|
| |
|
| Hybridization protocol |
Affymetrix GeneChip expression arrays (GeneChip Human Gene 2.0 ST Array) were hybridized for 16 h in a 45°C incubator, rotated at 60 rpm.
|
| Scan protocol |
Hybridized arrays were scanned on an Affymetrix GeneChip Scanner 3000 7G.
|
| Description |
Gene expression data from decidual cells treated with vehicle.
|
| Data processing |
Gene level differential expression analysis was performed using the Affymetrix Transcriptome Analysis Console (TAC) software version 1.0 using the default analytical pipeline, including the following algorithms: Tukey's Biweight averaging, unpaired single factor Analysis of Variance (ANOVA), and the Benjamini-Hochberg step-up false discovery rate (FDR)-controlling procedure.
|
| |
|
| Submission date |
Jun 04, 2014 |
| Last update date |
Jan 01, 2016 |
| Contact name |
Douglas A Kniss |
| E-mail(s) |
kniss.1@osu.edu
|
| Phone |
614-293-4496
|
| Organization name |
Ohio State University
|
| Department |
Obstetrics and Gynecology
|
| Lab |
Perinatal Research Lab
|
| Street address |
395 W. 12th Ave
|
| City |
Columbus |
| State/province |
Ohio |
| ZIP/Postal code |
43210 |
| Country |
USA |
| |
|
| Platform ID |
GPL16686 |
| Series (1) |
| GSE58220 |
Expression data from primary term human decidual cells treated with interleukin-1-beta for 6 hours. |
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