Squirrel tissues were rapidly dissected and frozen on liquid nitrogen. For each tissue sample total RNA was extracted using Trizol (Invitrogen). Fluorescently labelled cDNA was synthesized using amino-allyl adducts coupled to Cy3 and Cy5 fluors (www.microarrays.org) and compared to a reference RNA by hybridization to two arrays with reversal of the labeled fluorophores. The reference RNA pool was prepared for each tissue by pooling RNA samples isolated from animals sampled under all conditions. Microarrays were hybridized overnight at 65Ž°C, washed, scanned, and the images scanned and quantified (GenePix 4000A, Axon Instruments, CA). Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 3000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. Without background correction, the log base 10 expression ratios of the median pixel intensities were established, and the spatial and intensity based trends in the data were removed by Lowess normalization (GeneSpring, Silicon Genetics). Data for each pair of fluor-reversed slides were combined by averaging the ratios derived from the two measurements. The expression of each cDNA was then normalized to the median ratio observed for that transcript in the summer animals.
The hibernating phenotype assessed through transcript screening
Data table header descriptions
ID_REF
VALUE
log ratio (log2 of PRE_VALUE)
Flags
Denotes which features met GenePix 3.0 flagging criterion. P means present. A means undetected. Neither P or A means spot was manually flagged by eye as being poor quality - generally dust or other artifact on slide surface.
