|
| Status |
Public on Feb 11, 2007 |
| Title |
3_hypothalamus_time_0_#119 |
| Sample type |
RNA |
| |
|
| Source name |
hypothalamus; sacrificed at lights on at 7 AM; this is “time 0”
|
| Organism |
Mus musculus |
| Characteristics |
Strain: C57/BL6/J
Gender: male
Age: 10 weeks of age ±1 week
Tissue: hypothalamus
|
| Biomaterial provider |
Miroslaw (Mirek) Mackiewicz, Ph.D.; University of Pennsylvania School of Medicine
|
| Treatment protocol |
Mice were sacrificed at lights on (7AM).
|
| Growth protocol |
Experiments were performed on male mice (C57/BL6/J), 10 weeks of age ±1 week. Animals were housed in a light/dark cycle of 12 hrs, in a pathogen free, temperature- and humidity-controlled room (22ºC and 45-55%, respectively) with water available ad libitum. Food was accessible for 12 hrs only during the active period. Animals were subjected to 14 days of acclimatization during which a nighttime feeding pattern was established. This was done to avoid differential food intake between mice that were subsequently sleep deprived during the lights on period and those allowed to sleep.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mice were sacrificed by cervical dislocation. Brain sectioning was performed according to the mouse brain atlas of Franklin and Paxinos, and broadly defined regions and zones of the hypothalamus were sampled. RNA was isolated with Trizol (Invitrogen) and further purified using RNeasy columns (Qiagen) as per the manufacturer’s instructions.
|
| Label |
biotin/streptavidin
|
| Label protocol |
Target preparation were performed as described by the Affymetrix GeneChip Expression Analysis Technical Manual (http://www.affymetrix.com), and were conducted at the University of Pennsylvania Microarray Core Facility. Briefly, 5 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.
|
| |
|
| Hybridization protocol |
Hybridization and post-hybridization procedures were performed as described by the Affymetrix GeneChip Expression Analysis Technical Manual (http://www.affymetrix.com), and were conducted at the University of Pennsylvania Microarray Core Facility. The cRNA products were fragmented to 200 nucleotides or less, heated at 99ºC for 5 min and hybridized for 16 h at 45ºC. 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.
|
| 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.
|
| Description |
none
|
| Data processing |
Affymetrix Microarray Suite 5.0 was used to quantitate expression levels for targeted genes; default values provided by Affymetrix were applied to all analysis parameters. Border pixels were removed, and the average intensity of pixels within the 75th percentile was computed for each probe. The average of the lowest 2% of probe intensities occurring in each of 16 microarray sectors was set as background and subtracted from all features in that sector. Probe pairs were scored positive or negative for detection of the targeted sequence by comparing signals from the perfect match and mismatch probe features. The number of probe pairs meeting the default discrimination threshold (tau = 0.015) was used to assign a call of absent, present or marginal for each assayed gene, and a p-value was calculated to reflect confidence in the detection call. A weighted mean of probe fluorescence (corrected for nonspecific signal by subtracting the mismatch probe value) was calculated using the One-step Tukey's Biweight Estimate. This Signal value, a relative measure of the expression level, was computed for each assayed gene. Global scaling was applied to allow comparison of gene Signals across multiple microarrays: after exclusion of the highest and lowest 2%, the average total chip Signal was calculated and used to determine what scaling factor was required to adjust the chip average to an arbitrary target of 150. All Signal values from one microarray were then multiplied by the appropriate scaling factor.
|
| |
|
| Submission date |
Dec 11, 2006 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Miroslaw Mackiewicz |
| E-mail(s) |
[email protected]
|
| Phone |
(215) 746-4805
|
| Fax |
(215) 746-4814
|
| Organization name |
University of Pennsylvania
|
| Department |
Medicine
|
| Lab |
Division of Sleep Medicine
|
| Street address |
2124 TRL 125S 31st Street
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104-3403 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE6514 |
Gene expression in the mouse brain during spontaneous sleep and prolonged wakefulness |
|
| Relations |
| Reanalyzed by |
GSE119085 |
