Genomic profiling of aged rat skeletal muscle. Rattus norvegicus

a genomic profile of aged rat sceletal muscle with diffrent degrees of sensorimotor disturbances. Keywords: age skeletal muscle sensorimotor disturbances Overall design: Experimental animals Male Sprauge-Dawley rats (strain Bkl; Harlan Sprauge-Dawley, Houston, TX), including a total of five adults (4 months old) and seven aged rats (30 months) were used. The animals were delivered by a local breeder (B and K, Stockholm, Sweden) at 2 months of age and were kept thereafter under standardized barrier-breeding conditions at our department (12-hour light/12-hour dark cycle) with free access to water and food (R70 with reduced protein content: Lactamin, Vadstena, Sweden). All experiments were approved by the Local Ethical Committee (Stockholm’s Norra Djurförsöksetiska Nämnd; project no. N54/00). Stage ranking of animal Under these conditions the median survival time is 30 months (plus/minus 2 months across cohorts), and based on this the 30-month-old rats are defined as aged. All aged rats used in this study showed signs of behavioral sensorimotor disturbances, defined by using two parameters as a guide to select a stage, namely: (a) is a limb weight bearing? (b) does a limb show a complete gait cycle coordinated with the other limb(s)? The animals were ranked in stage 0-III: Stage 0 animals show no signs of insufficiency concerning these two variables; Stage I animals show some signs, including a lowering of the trunk towards the supportive surface and minor signs of gait cycle aberrations; Stage II animals show clear gait cycle aberrations, often involving a poorer coordination between limbs. Body weight bearing is often clearly incapacitated in at least one limb; Stage III animals show severe gait cycle aberrations, often involving a poorer coordination between limbs. Only minor efforts to initiate a gait cycle may. Analysis of chips GenePix Pro software (Axon Instruments, CA) was used for the analysis of the image. The signal of each spot was calculated as the average intensity of the spot minus the background. Spots with intensity that were at least 1.6 times above the background were included in the study. Dye swaps were used in one of the chips in each group of runs. Normalization between the two fluorescent images was performed, as described previously [Ross DT et al., 2000], by applying a scaling factor to all intensities measured for the Cy5 channel so that the mean log (Cy5/Cy3) for the subset of spots that were not flagged was 0. Expression ratios calculated as Cy5/Cy3 signals was normalized using the “LOWESS” method in the SMA (Statistical Analysis of Macroarrays) package [Dudoit S et al., 2002]; [Yang YH et al., 2002] SMA is an add-on library written in the public domain statistical language R [Ihaka R et al., 1996]. The LOWESS (locally Weighted Scatter Plot Smoother) algorithm performs a local fit to the data in an intensity-dependent manner. The intensity value for each spot is normalized based on data distribution in the immediate neighborhood of the spot’s intensity. Genes with missing data in more than one of the three triplicates of the studied cases were excluded. The significance of the expression ratios of the aged animals in the different stage groups was estimated using the SAM software [Tusher VG et al., 2001]. SAM is a statistical technique for finding significantly regulated genes in a set of microarray experiments. For each gene, i, in the array SAM compute the T-statistics di, a score derived from changes of gene expression in relation to the standard deviation of repeated measurements for that gene. A threshold can be set based on di to identify potentially significant changes in gene expression. The threshold can be adjusted based on an associated false discovery rate (FDR) value: the percentage of genes expected to be false identified as differentially expressed when certain threshold (d value) is set. To each of the genes in the array a q-value was assigned. This value is similar to the commonly used p-value and measures the lowest FDR at which the gene is called significant. In this study, initially genes with a q-value of more than 5% in either if the stage groups in the same gene were excluded for further analysis.