Title: Affymetrix CEL analysis. Description:
Affymetrix Generic Hybridization
nucleic acid labeling protocol
Labeling of samples was carried out according to the manufacturer’s standard protocol
Background correction,quantile normalization, and gene expression analysis were performed using RMA (Bolstad, B.M., Irizarry R. A., Astrand, M., and Speed, T.P. (2003)A Comparison of Normalization Methods for High Density Oligonucleotide Array Data Based on Bias and Variance. Bioinformatics 19(2):185-193).
Male C57BL/6J mice (6 weeks old from Harlan laboratories) were entrained to a 12:12 hr light/dark cycle for 1 week in a light-tight cabinet. Animals were transferred into constant darkness for 39 hr dark adaptation prior to sacrifice. Circadian time (CT) was calculated based on the prior light-dark cycle; animals were culled in the dark and eyes removed, every 4 hr beginning at CT3. Xiphoid cartilage from 4-5 mice was pooled for each sample, with n=3 samples per time-point. Samples were snap-frozen in liquid nitrogen. All work was carried out in accordance with the 1986 Home Office Animal Procedures Act (UK) and following local ethical review, or approved by the Murdoch Childrens Institute Animal Ethics Committee (Approval #605). Mice were maintained at 20-22oC, with standard rodent chow available ad libitum.
(Parameters: time unit = seconds, temperature unit = C)
Xiphoid cartilage from 4-5 mice was pooled for each sample, with n=3 samples per time-point. Samples were snap-frozen in liquid nitrogen. Frozen tissue samples were homogenised in RLT-buffer using a Mikro-Dismembrator S [Satorius Stedim Biotech]. RNA extractions were carried out using RNeasy Fibrous Tissue Mini/Micro kits [Qiagen] with on-column DNAse digestion, according to instructions.
(Parameters: Extracted product = total_RNA, Amplification = none)