|
| Status |
Public on Jul 10, 2010 |
| Title |
follicular cells_competant/incompetant_rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Follicular cells collected at the same time as the competant oocyte
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: follicular cells
competancy status: competant
|
| Treatment protocol |
Follicular cells from oocytes that resulted in a 100% pregnancy success rate were consider as competant cells and follicular cells from oocytes that resulted in a transferred embryo with unsuccessful pregnancy were considered as incompetant cells.
|
| Growth protocol |
Following ovarian stimulation, follicular fluid, FCs and oocytes from individual follicles were collected by ultrasoundguided follicular aspiration using a double lumen needle. The oocytes and surrounding cumulus cells were removed for IVF treatment.The remaining follicular fluid was centrifuged at 800g for 10 min at room temperature to isolate the FCs containing mural granulosa cells, for each individual follicle. The resulting pellet was suspended in 500 ml of phosphate-buffered saline solution at 48C and was transferred into a cryovial. After centrifugation at 2000g for 1 min at room temperature, the supernatant was removed and cells were rapidly frozen and stored in liquid nitrogen until RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from follicular cells was extracted with 1 ml of Trizol reagent (Invitrogen) following the manufacturer’s protocol. RNA was then further purified using the RNeasy total RNA clean-up protocol with DNAse treatment (Qiagen). The concentration and integrity of the RNA samples were assessed using a 2100 Bioanalyzer (Agilent)
|
| Label |
Alexa 555,Alexa 647
|
| Label protocol |
Total RNA of follicular cells was amplified using the RiboAmpT7 RNA Amplification kit (Molecular Devices) according to the manufacturer’s instructions. 2 ug of the aRNA obtained was labelled with the ULS aRNA Fluorescent Labelling Kit (Kreatech Biotechnology) linked to Alexa 555 and 647.
|
| |
|
| Channel 2 |
| Source name |
Follicular cells collected at the same time as the incompetant oocyte
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: follicular cells
competancy status: incompetant
|
| Treatment protocol |
Follicular cells from oocytes that resulted in a 100% pregnancy success rate were consider as competant cells and follicular cells from oocytes that resulted in a transferred embryo with unsuccessful pregnancy were considered as incompetant cells.
|
| Growth protocol |
Following ovarian stimulation, follicular fluid, FCs and oocytes from individual follicles were collected by ultrasoundguided follicular aspiration using a double lumen needle. The oocytes and surrounding cumulus cells were removed for IVF treatment.The remaining follicular fluid was centrifuged at 800g for 10 min at room temperature to isolate the FCs containing mural granulosa cells, for each individual follicle. The resulting pellet was suspended in 500 ml of phosphate-buffered saline solution at 48C and was transferred into a cryovial. After centrifugation at 2000g for 1 min at room temperature, the supernatant was removed and cells were rapidly frozen and stored in liquid nitrogen until RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from follicular cells was extracted with 1 ml of Trizol reagent (Invitrogen) following the manufacturer’s protocol. RNA was then further purified using the RNeasy total RNA clean-up protocol with DNAse treatment (Qiagen). The concentration and integrity of the RNA samples were assessed using a 2100 Bioanalyzer (Agilent)
|
| Label |
Alexa 555,Alexa 647
|
| Label protocol |
Total RNA of follicular cells was amplified using the RiboAmpT7 RNA Amplification kit (Molecular Devices) according to the manufacturer’s instructions. 2 ug of the aRNA obtained was labelled with the ULS aRNA Fluorescent Labelling Kit (Kreatech Biotechnology) linked to Alexa 555 and 647.
|
| |
|
| |
| Hybridization protocol |
Slides were hybridized overnight at 50°C with labelled purified probes using the SlideHyb #1 buffer (Ambion). Hybridizations were performed in a SlideBooster (The Gel Company). Slides were then washed twice with 2X SSC/0.5% SDS for 15 min at 50°C and twice with 0.5X SCC/0.5% SDS for 15 min at 50°C. They were dried by centrifugation at room temperature at 1200g.
|
| Scan protocol |
Slides were scanned using the VersArray ChipReader System (Bio-Rad) and analyzed using the ChipReader and ArrayPro Analyzer software (Media Cybernetics).
|
| Data processing |
Data analysis was carried out using ArrayPro Analyzer software (Media Cybernetics), version 4.1 . The treat-mean values from each channel were corrected for background, log2 transformed and normalized using the LOESS algorithm to remove intensity dependent effects within the calculated values. Normalised values were used to calculate the fluorescence ratios from two experimental repeats with all replicates combined. Data was compiled from two dye-swap replicate.
|
| |
|
| Submission date |
Jul 09, 2010 |
| Last update date |
Jul 09, 2010 |
| Contact name |
Marc-André Sirard |
| E-mail(s) |
Marc-Andre.Sirard@fsaa.ulaval.ca
|
| Organization name |
Université Laval
|
| Department |
Sciences Animales
|
| Street address |
Offfice 2732, 2440 Hochelaga Blvd.
|
| City |
Québec City |
| State/province |
Quebec |
| ZIP/Postal code |
G1V 0A6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL10668 |
| Series (1) |
| GSE22869 |
Identification of follicular marker genes as pregnancy predictors for human IVF |
|
