They were then exposed to a 30 minute light pulse (2400lux) or a dark pulse (sham) starting one hour after lights off (Zeitgeber time (ZT) 13). Immediately after treatment, mice were euthanized via cervical dislocation. Dark pulse mice were euthanized under dim red light, and both light and dark pulse animals’ eyes were dissected out immediately to avoid excess exposure to light. The mice were then decapitated and the brains were quickly removed and frozen in isopentane cooled in dry ice. Brains were stored at -70?C until ready for further processing. Brains were cut into 12 µm thick sections on a cryostat and directly mounted onto glass slides. Sections were stained using a quick protocol to allow for visual identification of the suprachiasmatic nucleus. First the sections were fixed in a 75% EtOH solution for 30 seconds, rinsed in water to remove excess EtOH from the slide, and then immersed in Hematoxylin for 90 seconds. Slides were then washed in molecular biology grade water. The slides then were taken through an alcohol dehydration series of 75%, 95% and 100% EtOH for 30 seconds each, followed by immersion in xylenes for 5 minutes. The slides were removed from the xylene, and once the remaining xylenes had evaporated the slides were placed into a laser capture microscope (Arcturus) and the SCN was identified and captured into CapSure® HS LCM Caps (Molecular Devices). For microarrays and validation of microarrays by quantitative real-time PCR (qRT-PCR), 6 consecutive SCN sections were captured from each mouse. Samples from three mice were then pooled together within the same treatment, so in total there were 18 SCN sections pooled together.
Growth protocol
Adult male C57BL/6 mice were individually housed in a 14:10 light/dark cycle in their experimental room and cage for at least two weeks prior to the experiment.
Extracted molecule
total RNA
Extraction protocol
The pooled samples were purified using an RNA purification kit (Picopure from Molecular Devices) including a DNase treatment. A 1 µl aliquot of each sample was removed and processed on an Agilent Bioanalyzer 2100 using the Agilent Lab-on-a-Chip Picochip RNA kit. Only samples with RNA integrity numbers about 6.8 were processed further. RNA was stored at -70?C until ready to proceed. 1 ng of total RNA was then processed through two rounds of linear amplification using Riboamp HS amplification kits (Molecular Devices).
Label
biotin
Label protocol
Following the 2nd round of linear amplification, the total amount of cDNA was biotinylated according to the standard Affymetrix protocol (Expression Analysis Technical Manual, 2004, Affymetrix). 15 ug of labeled cRNA was fragmented, then 10 ug was hybridized to Affymetrix 430A 2.0 Genechips according to standard Affymetrix protocols (Expression Analysis Technical Manual, 2004, Affymetix)
Hybridization protocol
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on a GeneChip Mouse 430A 2.0 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
Scan protocol
Genechips were scanned using the Affymetrix Scanner 3000
Description
Gene Expression data from the suprachiasmatic nucleus of an animal euthanized 1.5 hours after lights off.
Data processing
The data were analyzed with GeneChip Operating Systtem 1.1 using the MAS 5.0 algorithm, using Affymetrix default analysis settings and global scaling as normalization method, using the default normalization target setting of 500.
