|
| Status |
Public on Apr 25, 2011 |
| Title |
Fem-E13_SRY_biological rep2 |
| Sample type |
RNA |
| |
|
| Source name |
E13 ovarian cell sub-culture, expression plasmid pCMV-myc over-expressing Sry gene, 6 days
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague-Dawley
gender: female
tissue: ovary
developmental stage: embryonic
age: E13
sub-cultured: less than 12 times
treatment: Sry overexpression
treatment time: 6 days
hybridization date: 6-23-10
|
| Treatment protocol |
Cells between sub 8 and 12 were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Four µg expression plasmid was mixed with 10 µl Lipofectamine 2000 in 500 µl Opti-MEM medium (Invitrogen) for each well of a 6-well plate. This 500 µl mix was added to each well containing ~90% confluent cultured E13 testis cells in 2 ml Ham’s F-12 medium without antibiotics and incubated at 32oC for 24 hours. After 24 hours, medium was aspirated from cells and replaced with 1ml Ham’s F-12 with 10% serum. Cells were incubated 6-7 days. In the end, 1 ml of Trizol was added and cells were collected for RNA extraction for microarray analysis.
|
| Growth protocol |
Male or female E13 embryos (14 to 18 tail somite-stage) were collected from timed-pregnant females. Gonads from E13 animals were dissected and each pair of gonads from individual animals was placed into one well of a 24-well plate with 500 µl Ham F-12 medium until embryos could be sexed. The gonads were then pooled and digested with trypsin (2.5%) and collagenase (1mg/ml type I) plus DNase (3mg/ml) to disassociate the cells. All the cells from the digested testes or ovaries were then cultured on 100 mm plates in Ham’s F-12 with 10% bovine calf serum (Sigma). Cells initially multiplied well in culture and were split two times (1:2) as they reached confluence, at which point cell division slowed considerably. Cells were maintained in culture, changing medium every three days, until growth plaques were observed at approximately one month. These growth plaques were then collected for further propagation and frozen stocks were prepared for subsequent cell splitting such that cells could be maintained at fewer than 12 subcultures.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from each of 3 well/plate separately after scraping cells from well bottom in 1 ml Trizol™ reagent (Sigma-Aldritch, USA), according to the manufacturer’s instructions.
|
| Label |
biotin
|
| Label protocol |
Biotin-labeled ssDNA were prepared according to the standard Affymetrix protocol from at least 300 ng total RNA (GeneChip Whole Transcript (WT) Sense Target Labeling Assay Manual, 2005-2209, Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, ssDNA were hybridized on the Affymetrix Rat Gene 1.0 ST Array according to the standard Affymetrix protocol. Chips were washed and stained in the Affymetrix Fluidics Station 4500.
|
| Scan protocol |
GeneChips were scanned using Affymetrix GeneChip Scanner 3000.
|
| Description |
F-E13_SRY-3
Gene expression data from E13 ovarian cell sub-culture incubated for 6 days in the presence of 2 ug expression plasmid pCMV-myc over-expressing rat Sry gene.
|
| Data processing |
The data (all 18 CEL files together) were analyzed with Partek Genomic Suite 6.5 beta software (Partek Inc., St. Louis, MO) using RMA, GC-content adjusted pre-processing algorithm for background correction, quintile normalization, median polish methods for probesets summarization, and log values of probes signals using base 2. Hybridization date batch effect was removed.
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| |
|
| Submission date |
Oct 12, 2010 |
| Last update date |
Apr 25, 2011 |
| Contact name |
Michael K Skinner |
| E-mail(s) |
[email protected]
|
| Organization name |
WSU
|
| Department |
SBS
|
| Street address |
Abelson 507
|
| City |
Pullman |
| State/province |
WA |
| ZIP/Postal code |
99163 |
| Country |
USA |
| |
|
| Platform ID |
GPL6247 |
| Series (1) |
| GSE24666 |
Basic helix-loop-helix transcription factor Tcf21 is downstream target of male sex-determining gene SRY |
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