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| Status |
Public on Mar 20, 2013 |
| Title |
Pb2-1 |
| Sample type |
RNA |
| |
|
| Source name |
su(Hw)Pb/2 virgin female ovaries dissected 4-6 hours post-eclosion
|
| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: ovary
genotype: y[2] w[67c23] ct[6] f[1]; su(Hw) [e04061]/su(Hw) [2]
age: 4-6 hours post-eclosion
|
| Treatment protocol |
Ovaries were dissected in ice-cold PBS and stored at -80C until RNA isolation
|
| Growth protocol |
Flies were raised at 25ºC, 70% humidity on standard cornmeal/agar medium supplemented with yeast.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from approximately 100 pairs of frozen ovaries per biological replicate using TRIzol reagent (Invitrogen), treated with DNaseI (Qiagen DNaseI) and purified using RNeasy columns (Qiagen) according to the manufacturer instructions.
|
| Label |
biotin
|
| Label protocol |
Fifty nanograms of total RNA was converted to cDNA using the Ovation RNA Amplification System v2 (NuGEN). The cDNA product was purified using QIAquick PCR Purification column (Qiagen) and 3.75 µg were fragmented to an average size of 85 bases and biotin labeled using the FL-Ovation cDNA Biotin Module (NuGEN) per the manufacturer’s protocol.
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| |
|
| Hybridization protocol |
Biotin-labeled cDNA was mixed with Affymetrix hybridization buffer, placed onto Drosophila 2.0 arrays (Cat #900532), and incubated at 45º C for 18 h with 60 rpm rotation in Affymetrix Model 640 GeneChip Hybridization Oven. Following hybridization, arrays were washed and stained with streptavidin-phycoerythrin (Molecular Probes). Signal was amplified with anti-streptavidin antibody (Vector Laboratories), followed by staining with streptavidin-phycoerythrin (Molecular Probes) using the Affymetrix Model 450 Fluidics Station (Affymetrix).
|
| Scan protocol |
Arrays were scanned with the Affymetrix Model 3000 scanner with 7G upgrade.
|
| Description |
Gene expression data from dissected ovaries 4-6 hours old, i.e. before stage 9 egg chambers
ovaries at 4-6 hours post-eclosion, replicate 1
|
| Data processing |
The data was analyzed with Partek 6.5 Suite using GCRMA analyses settings (GCRMA background correction, quantile normalization, median polish probeset summarization)
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| |
|
| Submission date |
Mar 19, 2013 |
| Last update date |
Mar 20, 2013 |
| Contact name |
Alexey A Soshnev |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Iowa
|
| Department |
Biochemistry
|
| Lab |
Geyer
|
| Street address |
3159C MERF 375 Newton Rd
|
| City |
Iowa City |
| State/province |
Iowa |
| ZIP/Postal code |
52246 |
| Country |
USA |
| |
|
| Platform ID |
GPL1322 |
| Series (1) |
| GSE45286 |
Expression analyses in Drosophila ovaries |
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