|
| Status |
Public on Sep 11, 2007 |
| Title |
TS-108 |
| Sample type |
RNA |
| |
|
| Source name |
Control Drosophila melanogaster larvae not infected by wasps, comparable to 9 to 12 hours post-infection timepoint, replicate 2.
|
| Organism |
Drosophila melanogaster |
| Characteristics |
40 second-instar (72 hours old at 22 degrees C) uninfected Drosophila melanogaster larvae (strain Oregon R) were collected for tissue homogenization and RNA extraction in the same manner as the infected larvae collected 9 to 12 hours post-infection.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the standard Trizol method (Gibco), and was purified using RNeasy spin columns (Qiagen).
|
| Label |
biotin
|
| Label protocol |
RNA processing and hybridization protocols are available at www.affymetrix.com (Santa Clara, CA) and in the Genechip Expression Analysis Technical Manual. Two micrograms of total RNA were converted into double-stranded cDNA by using the GeneChip Expression 3’-Amplification Reagents One-Cycle cDNA Synthesis Kit with the T7-(dT)24-primer [sequence 5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3’]. The cDNA was purified as per protocol by using the GeneChip Sample Cleanup Modules. Purified cDNA was then used for in vitro transcription using the GeneChip Expression 3’-Amplification Reagents for IVT Labeling. Biotin-labeled cRNA was purified by GeneChip Sample Cleanup Modules and then randomly chemically fragmented to approximately 35 - 200 bp pieces.
|
| |
|
| Hybridization protocol |
Each fragmented cRNA sample was hybridized to the Affymetrix Drosophila Genome microarray for 16 hours at 60 rpm at 45°C. The microarray was washed and stained on the Affymetrix Fluidics Station 450 according to manufacture’s protocol. After washing out the nonhybridized material the array was incubated with phycoerythrin-streptavidin (SAPE, Molecular Probes, Eugene, OR). The signal intensity was amplified by second staining with biotin-labeled antistreptavidin antibody followed by SAPE staining.
|
| Scan protocol |
Fluorescent images were read using Affymetrix GeneChip Scanner 3000. The microarrays were processed on the fluidics station under the control of the GeneChip Operating software (GCOS) and read according to the standard Affymetrix protocol. The signal, which represents the intensity of each gene, was extracted from the image. The target intensity value from each chip was scaled to 500. All RNA samples passed the quality control and ratio of 3’ to 5’ less than three.
|
| Description |
Labeling, hybridization, and scanning protcols were carried out at the Weill Medical College Microarray Core Facility of Cornell University.
|
| Data processing |
The GeneTraffic (Stratagene) software package was used for microarray data processing. Probe intensities were normalized using Robust Multi-Chip Analysis, using the three control replicates from each time point as the designated baseline.
|
| |
|
| Submission date |
Sep 02, 2007 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Todd A Schlenke |
| E-mail(s) |
tschlen@emory.edu
|
| Phone |
404-727-0817
|
| Fax |
404-727-2880
|
| Organization name |
Emory University
|
| Department |
Department of Biology
|
| Lab |
RRC room 1081
|
| Street address |
1510 Clifton Road NE
|
| City |
Atlanta |
| State/province |
GA |
| ZIP/Postal code |
30322 |
| Country |
USA |
| |
|
| Platform ID |
GPL1322 |
| Series (1) |
| GSE8938 |
Contrasting infection strategies in generalist and specialist wasp parasitoids of Drosophila melanogaster. |
|
| Relations |
| Reanalyzed by |
GSE119084 |
