|
| Status |
Public on Feb 06, 2013 |
| Title |
Em19-5A |
| Sample type |
RNA |
| |
|
| Source name |
NESC
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: endometrial tissue
cell type: NESC
|
| Treatment protocol |
ECSCs and NESCs isolated and cultured as described above were subjected to miRNA microarray analysis. Cells subjected to microarray did not undergo any stimulation during culture.
|
| Growth protocol |
Endometriotic tissues were obtained from premenopausal patients who had undergone salpingo-oophorectomy or evisceration for ovarian endometriotic cysts. Normal endometrial tissues were obtained from premenopausal patients who had undergone hysterectomies for subserousal leiomyoma and had no evidence of endometriosis. None of the patients had received any hormonal treatments for at least 2 years prior to operation. All the specimens were diagnosed as being in the mid- to late-proliferative phases according to the pathological observation and/or the menstrual cycles. ECSCs and NESCs were isolated from ovarian endometriotic tissues by enzymatic digestion with collagenase as previously described (Nishida et al., J Clin Endocrinol Metab 2004; 89: 5094-5100). Isolated ECSCs and NESCs were cultured in DMEM supplemented with 100 IU/ml of penicillin, 50 mg/ml of streptomycin, and 10% heat-inactivated fetal bovine serum at 37°C in 5% CO2 in air. ECSCs and NESCs in the monolayer culture after the third passage were >99% pure as determined by immunocytochemical staining with antibodies to vimentin, CD10, cytokeratin, factor VIII, and leukocyte common antigen, and were used for the following experiments (Nishida et al., J Clin Endocrinol Metab 2004; 89: 5094-5100).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from cultured ECSCs and NESCs was extracted with a miRNeasy Mini kit (QIAGEN) following the manufacturer's instructions. RNA was quantified using a NanoDrop-1000 spectrophotometer.
|
| Label |
Cy3
|
| Label protocol |
One hundred ng of total RNA was labeled with Cy3 using a miRNA labeling and hybridization kit (Agilent Technologies) according to the manufacturer’s instructions.
|
| |
|
| Hybridization protocol |
After Cy3-labeling, RNA was dried up and re-solved with 18 ul RNase-free water. And then, 4.5 ul of 10x Gene expression blocking agent and 22.5 ul of 2x hybridization buffer HI-RPM were added to the RNA sample, and the mixture hybridized to Agilent Human miRNA Microarrays (G4470C, Human Rel. 12.0, Agilent Technologies) for 20 hours at 55°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 5 minute at room temperature with GE Wash Buffer 1 (Agilent Technologies) and 5 minute with 37°C GE Wash buffer 2 (Agilent Technologies).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565BA) using one color scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 5% and 100%, extended dynamic range scan mode).
|
| Description |
miRNA expression in primary cultured NESCs
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5.3.1 (Agilent Technologies) using default parameters (protocol miRNA-v1_95_May07 and Grid: 021827_D_20081121) to obtain background subtracted Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Aug 17, 2012 |
| Last update date |
Feb 06, 2013 |
| Contact name |
Kaei Nasu |
| E-mail(s) |
nasu@oita-u.ac.jp
|
| Organization name |
Oita University, Faculty of Medicine
|
| Department |
Obstetrics and Gynecology
|
| Street address |
Idaigaoka 1-1, Hasama-machi
|
| City |
Yufu-shi |
| State/province |
Oita |
| ZIP/Postal code |
879-5593 |
| Country |
Japan |
| |
|
| Platform ID |
GPL14767 |
| Series (1) |
| GSE40186 |
miRNA expression profiles in endometriotic cyst stromal cells (ECSCs). |
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