|
| Status |
Public on Feb 27, 2006 |
| Title |
Brain_Deprived_2hr_1 |
| Sample type |
RNA |
| |
|
| Source name |
Whole Brain_Deprived_2hr
|
| Organism |
Drosophila melanogaster |
| Characteristics |
Strain: Canton-S
Gender: Female
Age: 5 days
Tissue: Whole Brain
Time of Sacrifice: ZT16
Condition: Sleep Deprived
|
| Treatment protocol |
Animals were sacrificed by immersion in >95°C distilled water, removed from water and stored at -70 ° C until tissue dissection performed within 24 hours of sacrifice. Brains were removed from the head and cleared of surrounding tissues using forceps under a dissecting microscope under ice cold insect ringers (130 mM NaCl, 4.7 mM KCl, 1.9 mM CaCl2) and immediately placed in Trizol® Reagent (U.S.Patent No.5,346,994).
|
| Growth protocol |
Females were collected as newly eclosed virgins and kept in populations of 50 flies at 25°C under 12 hour light: 12 hour dark conditions in vials containing standard cornmeal media until time of sacrifice.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the pools of 139 (min) to 193 (max) brains using TRIZOL Reagent (U.S.Patent No.5,346,994) following the manufactrurers instructions (Invitrogen)
|
| Label |
Biotin
|
| Label protocol |
All protocols were conducted as described in the Affymetrix GeneChip Expression Analysis Technical Manual. Briefly, 5.0 ug of total RNA was converted to first-strand cDNA using Superscript II reverse transcriptase primed by a poly(T) oligomer that incorporated the T7 promoter. Second-strand cDNA synthesis was followed by in vitro transcription for linear amplification of each transcript and incorporation of biotinylated CTP and UTP. Performed by the staff at the Penn Microarray Facility http://www.med.upenn.edu/microarr/index.htm
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| |
|
| Hybridization protocol |
The cRNA products were fragmented to 200 nucleotides or less, heated at 99ºC for 5 min and hybridized for 16 h at 45ºC to 1 microarray. The microarrays were then washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain. Performed by the staff at the Penn Microarray Facility http://www.med.upenn.edu/microarr/index.htm
|
| Scan protocol |
A confocal scanner was used to collect fluorescence signal at 3um resolution after excitation at 570 nm. The average signal from two sequential scans was calculated for each microarray feature. Performed by the staff at the Penn Microarray Facility http://www.med.upenn.edu/microarr/index.htm
|
| Description |
This is one of seven timepoints (2 Hour Deprivation) in a study the effects of sleep deprivation on gene expression in the Drosophila brain.
|
| Data processing |
Ten time groups were used in this study, each with three biological replicates. Seven time groups were used to determine differential gene expression between normal and sleep-deprived flies while three time groups were used as an experimental control to test for differential gene expression due to the effect of mechanical stimulation. Probe intensity data from all 30 arrays was read into the R software environment (http://www.R-project.org) directly from .CEL files using the R/AFFY package (Gautier et al., 2004). R/AFFY was also used to extract and manipulate probe level data to assess data quality and to create expression summary measures. Normalization was carried out using the robust multi-array average (RMA) method (Irizarry et al., 2003) on all probe intensity data sets together in order to form one expression measure for each gene on each array. Briefly, the RMA method adjusted the background of perfect match (PM) probes, applied a quantile normalization of the corrected PM values, and calculated final expression measures using the median polish algorithm.
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| |
|
| Submission date |
Feb 02, 2006 |
| Last update date |
Feb 23, 2006 |
| Contact name |
John E Zimmerman |
| E-mail(s) |
[email protected]
|
| Phone |
215-746-4804
|
| Fax |
215-746-4813
|
| URL |
http://www.uphs.upenn.edu/sleepctr/centerforsleep.htm
|
| Organization name |
University of Pennsylvania School of Medicine
|
| Department |
Center for Sleep & Respiratory Neurobiology
|
| Lab |
Dr. Allan I. Pack
|
| Street address |
125 South 31st Street
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL72 |
| Series (1) |
| GSE4174 |
Effect of Sleep Deprivation on the Whole Brain of Drosophila |
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