Hibernation is an energy saving adaptation that involves a profound suppression

Hibernation is an energy saving adaptation that involves a profound suppression of physical activity that can continue for 6-8 weeks in highly seasonal environments. torpor of hibernation that suggests induction of translation at different hibernation claims. The induction of protein biosynthesis likely contributes to attenuation of disuse muscle mass atrophy through the long term periods of immobility of hibernation. The lack of directional changes in genes of protein catabolic pathways does not support the importance of metabolic suppression for conserving muscle mass during winter season. Coordinated reduction of multiple genes involved in oxidation reduction and glucose rate of metabolism detected in both species is consistent with metabolic suppression and lower energy demand in skeletal muscle mass during inactivity of hibernation. (Shao was selected as a research gene for black bear skeletal muscle mass and for floor squirrel based on the stability of manifestation ideals across all samples from the microarray experiments and then tested by RT PCR. All samples showed similar manifestation ideals for the correspondent research genes with low Compound 401 standard deviation in multiple RT-PCR checks. Total RNA concentrations were measured having a NanoDrop ND-1000 spectrophotometer and cDNA was synthesized from 0.5 ��g of total RNA from each sample. The reverse transcription was carried out with MiltiScribeTM reverse transcriptase (Applied Biosystems) with oligo d(T)16 primer in 25 ��l reactions at 25�� C for 10 min 48 C for 30 min and at 95�� C for 5 min. The synthesized cDNA was diluted 4x with RNase-free water and 4 ��l of diluted cDNA was used in the 20 ��l-volume real-time PCR. Primers were designed with the Primer3 software (http://frodo.wi.mit.edu/primer3/) using the black carry and arctic floor squirrel EST sequences (Table S1). Real-time PCR was performed in triplicates with Power SYBR Green PCR Expert Blend (Applied Biosystems) on an ABI-7900 HT. Biking parameters were 50�� C for 2 min of incubation 95 C for 10 min of Taq activation and 40 cycles of 95�� C for 15 sec and 60�� C for 1 min. Settings with no template were arranged to exclude contamination and controls with no reverse transcriptase but all other components were taken to exclude nonspecific amplification from genomic DNA. Specificity of amplification was checked with the melting curve analysis and agarose gel electrophoresis. Four 10-collapse dilutions of a sample with combined cDNA from all samples were used for a standard curve for each primer arranged for calculating RT PCR effectiveness. We tested a difference in gene manifestation between hibernating and summer season active animals with P < 0.10 as cutoff according to Pfaffl (2001). We determined the fold-change in level of manifestation of a target gene relative to a research gene for each sample and then compared the Compound 401 ideals for each group using Student��s t-test as explained by Livak & Schmittgen (2001). Results Differentially indicated genes in black bears Signals from 9 93 of 9 600 (95%) probes across all samples showed median Compound 401 intensities that were above background. A total of 415 unique genes (4.6% of all genes with significant signals) were differentially indicated in the skeletal muscle tissue of bears sampled during hibernation compared with in bears sampled in summer time BAMBI (Table S2). All but three differentially indicated genes (was over-expressed during torpor of hibernation in both species (Table 1) which is consistent with elevated manifestation of this gene previously recognized by Compound 401 RNA-seq in muscle mass of torpid 13-lined floor squirrels (Hampton 2009; this study). Arrows show direction of gene … Table 3 Gene Ontology categories of biological processes significantly enriched with differentially indicated genes in the arctic floor squirrel skeletal muscle mass. Arrows indicate direction of gene manifestation changes in torpid animals. FDR is false discovery rate. … Apart from validation of the GO miner result we also used GSEA to test enrichment in selected gene units (Molecular Signature data foundation) known to be important for muscle mass homeostasis (Table 4). In both species gene units separately involved in three phases of translation (initiation elongation.