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| Status |
Public on Jun 01, 2016 |
| Title |
cumulus cells of PCOS patient_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
cumulus cells isolated from PCOS patient
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: cumulus cells
disease state: PCOS
|
| Treatment protocol |
All the PCOS participants included in the study were women undergoing in vitro fertilization.Ovarian stimulation and oocyte retrieval protocols were carried out as below.pituitary down-regulation was started with mid-luteal phase GnRH agonist (Diphereline 3.75mg, PharmaBiotech, Paris, France). Once adequate pituitary down-regulation was confirmed [absence of ovarian cysts>=18mm in diameter and serum estradiol (E2) levels of <50 pg/ml]. Controlled ovarian stimulation was conducted with recombinant FSH (Follitropin Alfa, Serono, London, UK). Human chorionic gonadotropin (hCG; 10,000 IU, Profasi; Serono Laboratories) was given in when two or more follicles were at least 18mm in diameter and the serum E2 levels were at least 300pg/ml per dominant follicle. Oocyte retrieval was performed 36h after hCG administration. Cumulus cells were separated from the oocyte with strippers mechanically and washed twice in cold phosphate buffer saline (Dubelcco’s medium) (Gibco, invitrogen, Paris, France) then centrifuged at 200g for 10 min. The supernatant was removed and the pellet was resuspended in RLT buffer of the Reasy Mini Kit.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression of cumulus cells in PCOS women
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Aug 21, 2015 |
| Last update date |
Jun 01, 2016 |
| Contact name |
Xin Huang |
| E-mail(s) |
[email protected]
|
| Organization name |
Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine
|
| Department |
Reproductive Medicine Centre
|
| Street address |
No.2699 Gaoke east road
|
| City |
Shanghai |
| State/province |
Shanghai |
| ZIP/Postal code |
210000 |
| Country |
China |
| |
|
| Platform ID |
GPL16543 |
| Series (1) |
| GSE72274 |
Altered microRNA profiles in cumulus cells isolated from PCOS patients |
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