At 12 weeks of age, lean C57BL/6 mice were fasted by removing food at 7:30 am and then dosed with vehicle (0.25% MC, 5% Tween-80, 0.02% SDS) or AMPK activators by oral gavage at around 9:30 am, and kept sedentary for the remainder of the study period. For the exercise arm, mice were put on a treadmill (Columbus Instruments International, OH) for exercise with speed set at 10 meter/min. Mice exercised for 30 min followed by 30 min rest and the total running distance was 1300 meters.
Growth protocol
Male lean C57BL/6 were purchased from Taconic (Hudson, NY) at 8 weeks of age and maintained on chow diet (Teklad 7012, Research Diets, Inc., New Brunswick, NJ) with free access to water in a 12 h light/12 h dark cycle.
Extracted molecule
total RNA
Extraction protocol
Samples are processed in parallel in 96-well plates to minimize potential variation. Reaction purification is achieved using magnetic binding beads for cDNA and Qiagen RNeasy kits for cRNA purification.
Label
biotin
Label protocol
The reverse transcription incorporates biotin moieties that are later labeled with phycoerythrin. After amplification, samples are put through a controlled fragmentation to improve hybridization sensitivity and consistency. The labeled molecules are biotinylated-cDNA.
Hybridization protocol
GeneChip microarrays are loaded with the fragmented target sample/hybridization buffer mix using standard manual techniques. Arrays are hybridized for 18 hours at 45C with vigorous mixing. Unbound sample is removed and staining is accomplished through the binding of streptavidin conjugated phycoerythrin to the hybridized target. Excess label is removed. Washing and staining steps are performed by the Affymetrix FS450 fluidics station using standard protocols.
Scan protocol
Arrays are scanned using a GeneChip Scanner 3000 7G with a 48 array autoloader. The scanner maintains the optimal temperature for the arrays prior to and during scanning.
Description
Mice were treated with a single dose of the short acting AMPK activator, SA2, at 3mpk and 6 hours post dose Heart was taken for RNA profiling.
Data processing
Gene expression data were processed using the Robust Multiarray Average (RMA) pipeline and one-way ANOVA analysis was performed to obtain fold change, raw p values, and False Discovery Rate Benjamini & Hochberg (FDR_BH) corrected p values using the Array Studio Software (Omicsoft Corporation, Cary, NC). Probe sets had to pass a pre-filter of Affymetrix MAS5 present call p-value < 0.05 in > 50% of the samples, per treatment group, to qualify for further analysis. Average fold changes between 1) exercise and sedentary (vehicle treated) or 2) compound and vehicle treatment groups (both sedentary) were generated with the sedentary vehicle group serving as baseline.
