|
| Status |
Public on May 02, 2013 |
| Title |
placental trophoblast_HLAG-/TROP2+_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
placenta, 8-12 week LMP
|
| Organism |
Homo sapiens |
| Characteristics |
cell source: primary human placenta from fetal demise
cell surface marker: TROP2 (TACSTD2)
|
| Treatment protocol |
hESC were cultured in irradiated MEF-conditioned medium supplemented with either 4ng/ml FGF2 or 10 ng/ml BMP4 (R&D Systems) under a standard gas atmosphere of humidified air/5% CO2 at 37°C.
|
| Growth protocol |
Human ES H1 (NIH code WA01) was maintained on irradiated mouse embryonic fibroblast (MEF) cells in 80% DMEM/F12 supplemented with 20% KnockOut serum replacement, 1 mM L-glutamine/0.1 mM 2-mercaptoethanol/1% nonessential amino acids (Life Technologies) and 4 ng/ml FGF2 . Placentas were collected from women undergoing surgery at 8-12 weeks of gestation by LMP (last menstrual period) due to fetal demise at University of Missouri Women‘s and Children’s Hospital. Trophoblast were isolated by mincing villous tissue and then incubating in dissociation solution (4.2mM MgSO4, 0.25 % trypsin, 100 kU/ml DNase I inDPBS) for 30 min at 37C. Cells were passed through a 100 µm cell filter, washed in Medium 199 and layered on a 40%/25% Percoll gradient. The TB layer was collected at the interface then washed and resuspended in PBS/ 0.5%BSA/ 2mM EDTA (PBS/BSA/EDTA).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Both placental cells and BMP4-treated hESC were incubated with an anti-HLA-G antibody, conjugated to phycoerythrin (PE) and an anti-TACSTD2 (TROP2) antibody, conjugated to biotin for 10 min at 4C. Cells were washed in PBS/BSA/EDTA (x 3), then incubated in a 1:5 dilution of anti-PE antibody -conjugated to magnetic beads (Miltenyi Biotec, Auburn CA) at 4C for 10 min to tag HLA-G+ cells.. Cells were then separated into HLA-G+ and – fractions by using MS columns and a MimiMACS Separator held by a Multistand (Miltenyi) to capture magnetic beads. HLA-G- cells that passed through the columns were incubated in a 1:5 dilution of streptavidin -conjugated magnetic beads (R&D Systems) at 4C for 10 min to tag HLAGHLA-G-/TACSTD2+ cells. A second MS column was used to collect the bead-bound (HLA-G-/TACSTD2+) cells. Total RNA was obtained using Ribopure (Ambion).
|
| Label |
Cy3
|
| Label protocol |
First and second strand cDNA was prepared from the total RNA samples. cRNA target was prepared from the DNA template and verified on the Bioanalyzer.
|
| |
|
| Hybridization protocol |
Purified cRNA (1 µg) was fragmented to uniform size and applied to Agilent 4x44K v2 microarrays (Design ID 026655, Agilent Technologies, Santa Clara, CA) in hybridization buffer. Arrays were hybridized at 65° C for 17 h on a rotating incubator and washed at 37° C for 1 min.
|
| Scan protocol |
Rinsed and dried arrays were scanned with an Agilent G2565 Microarray Scanner (Agilent Technologies, Santa Clara, CA) at 5 µm resolution.
|
| Description |
TROP2 placental 1
|
| Data processing |
Agilent Feature Extraction software was used to process the scanned images from arrays (gridding and feature intensity extraction) and the data generated for each probe on the array was analyzed with GeneSpring GX v7.3.1 software (Agilent Technologies, Santa Clara, CA). Values are normalized to 75th percentile intensity for each array (=1).
|
| |
|
| Submission date |
Nov 07, 2012 |
| Last update date |
May 02, 2013 |
| Contact name |
Laura Clamon Schulz |
| E-mail(s) |
SchulzL@missouri.edu
|
| Organization name |
University of Missouri
|
| Department |
Obstetrics, Gynecology and Women's Health
|
| Street address |
1 Hospital Dr.
|
| City |
Columbia |
| State/province |
MO |
| ZIP/Postal code |
65212 |
| Country |
USA |
| |
|
| Platform ID |
GPL13497 |
| Series (1) |
| GSE42112 |
Comparison of extravillous trophoblast cells derived from human embryonic stem cells and from first trimester human placentas |
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