|
| Status |
Public on Jun 26, 2014 |
| Title |
darkcd4_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
whole body, darkcd4 mutant
|
| Organism |
Drosophila melanogaster |
| Characteristics |
genotype/variation: w1118;darkcd4
developmental stage: day 5 adult
tissue: whole body
|
| Treatment protocol |
After adult eclosion, flies were collected within 1 day and kept them five days with free access to food and mating.
|
| Growth protocol |
Flies were kept at 25 °C on standard diet containing cornmeal, baker's yeast, and glucose in 60% humidity with light/dark (12h/12h) condition.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified from five adults for one sample using TRIzol and purified by RNeasy Plus Micro Kit (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 50 ng RNA using the Low Input Quick Amp Labeling Kit, One-Color (Agilent) according to the manufacturer's instructions, followed by RNeasy Mini Kit (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop 2000c Spectrophotometer.
|
| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/μg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 μl containing 1x Agilent fragmentation buffer and 2x Agilent GE blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 25 μl of 2x Agilent GE hybridization buffer HI-RPM was added to the fragmentation mixture and hybridized to SurePrint G3 custom microarray 8x60K (G4102A, #040871) 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.
|
| Scan protocol |
Slides were scanned immediately after washing on the SureScan Microarray Scanner using AgilentG3_GX_1Color_HighSensitivity.
|
| Description |
dark2
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 040871_D_F_20120511) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Data were normalized by Percentile Shift (at 75 % percentile) and subjected to baseline transformation (baseline to median of all samples) using GeneSpring GX.
|
| |
|
| Submission date |
Jun 11, 2013 |
| Last update date |
Jun 26, 2014 |
| Contact name |
Masayuki Miura |
| E-mail(s) |
miura@mol.f.u-tokyo.ac.jp
|
| Organization name |
the University of Tokyo, Grad. Sch. Pharm. Sci
|
| Department |
Genetics
|
| Street address |
7-3-1, Hongo
|
| City |
Bunkyo-ku |
| State/province |
Tokyo |
| ZIP/Postal code |
1130033 |
| Country |
Japan |
| |
|
| Platform ID |
GPL17277 |
| Series (1) |
| GSE47853 |
The gene expression changes in darkcd4 apoptosis-deficient mutant flies |
|
