Eight week old mice were housed individually with 'locked' running wheel. Food / water provided ad libitum; 12h light/dark cycles; temperature and humidity maintained at 19-21°C and 40-50%, respectively. After one week, the mice were anesthetized and subsequently euthanized, with 2-4% isofluorine. Body composition was analyzed prior to sacrificing, using the Lunar Piximus DEXA.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from nucleus accumbens, soleus, and EDL using the Qiagen miRNeasy kit (Qiagen, Valencia, CA), with inclusion of the on-column DNA treatment. RNA quantified using Nanodrop 1000 spectrophotometer and quality of RNA was determined using an Agilent 2100 Bioanalyzer.
Label
Cy3
Label protocol
total RNA diluted to 50ng/μl; prepared 3 dilutions of labeling spike-in. Prepared CIP Master Mix (10.8μl 10X calf intestinal phosphatase buffer, 29.7μl labeling spike-in, 13.5μl calf intestinal phosphatase; total volume for 24 reactions). Mixed 2μl diluted RNA + 2μl Master Mix, incubate at 37°C in heat block for 30min. Stored in -80 until further use. Denature by adding 2.8μl DMSO and incubating at 100° for 5-10min; quench on ice. Warmed 10x T4 Ligate buffer at 37° and then cooled to RT. Prepared ligation master mix (27μl T4 ligase buffer, 81μl Cy3, 13.5μl T4 RNA ligase; total volume for 24 reactions). 4.5μl ligation MM added to each sample; incubate at 16°° for 2 hours. Used micro bio-spin columns for labeled RNA purification. Concentrated samples using vacuum at 50°C for 1 hour.
Hybridization protocol
Dried samples resuspended in 17μl nuclease-free water and added 1μl 3rd dilution hyb spike in, add 4.5μl 10x blocking agent + 22.5μl 2x Hi-RPM hyb buffer to each sample. Incubate at 100°for 5 min, quench in ice water for 5 min. Loaded hybridization assembly according to manufactures directions. Hybridized for 20 hours at 55°C. Added Trition X to wash buffers; warmed buffer 2 overnight. Scanned slides immediately after washing using Agilent Scanner B, AgilentG3_miRNA setting for the 8X60k format. Resolution, 3 microns; extended range (using 16 bit TIFF setting); dye channel, green. Used GEML file 035430_D_F_20121221.
Scan protocol
Scanned slides immediately after washing using Agilent G2505C scanner, AgilentG3_miRNA setting for the 8X60k format. Resolution, 3 microns ; extended range (using 16 bit TIFF setting); dye channel, green.
Data processing
Used GEML file 035430_D_F_20121221 for feature extraction, along with miRNA_107_Sept09 protocol. Quality reports were viewed and heterogenous intensity and 5 arrays were discluded (blank sample names in metadata template). Agilent GeneSpring GX12.5 software was used to process arrays. For soleus and nucleus accumbens, probes with low values caused errors in statistical analysis (homogenous p-values and FC values for all 'significantly different' miRNAs). Throughcommunication with Agilent, low intensity probes were removed from the raw files and entity list were manually imported into GeneSpring for further analysis. Separate 'experiments' were created in GeneSpring for each tissue, thus the only variable is strain. Percentile shift normalization was used (setting at defult of 90%) and threshold raw signals to 1.0, baseline to median. Additionally, filtered on expression of normalized data, by percent (20-100%), 75% of values in any 1 out of 2 conditions included. Filtered on flags, including 'detected' and 'not detected', again, 75% of values in any 1 out of 2 conditions included. Statistical analysis done by moderated t-test, p value < 0.05 and FC > 2, with Benjamini-Hochberg multiple testing correction.
