gender: male strain: C57BL/6J age: 12 weeks tissue: triceps brachii
Treatment protocol
Adult mice were given a continuous infusion of saline or 20-hydroxyecdysone (5 mg/kg/day) for 5 or 15 days via subcutaneously implanted Alzet® osmotic pumps.
Growth protocol
Animals were individually housed in plastic cages in a temperature controlled room (22 °C) with 12:12 hour light-dark cycles and given ad libitum access to food (AIN-93G) and water.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from triceps (ca. 100 mg) by homogenization with 1 mL TriReagent (St. Louis, MO) for 3 min at 30Hz using a Tissuelyser II (Qiagen, Gaithersburg, MD). Total RNA was treated with DNase I and purified using an RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. Quantity and purity of RNA was determined using the NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA). RNA quality was determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Total RNA was pooled into groups containing three individual biological replicates (except for one pool of saline infused group B which had two replicates). Each pool contained an equal quantity of RNA (1 μg) from each individual animal. Three pools were analyzed per treatment using Illumina’s MouseWG-6 v2.0 Expression BeadChips (San Diego, CA).
Label
biotin
Label protocol
300 ng of high quality total RNA were primed with an oligo(dT) primer bearing a T7 promoter, and reverse transcribed in the first-strand cDNA synthesis using the Illumina® TotalPrep RNA Amplification Kit (Ambion, Inc., Austin, TX). Single stranded cDNA was then converted into double stranded cDNA according to the manufacturer’s instructions. The double-stranded cDNA was purified and served as a template in the 14-hour in vitro transcription (IVT) reaction. Prior to hybridization, the synthesized biotin labeled cRNA was cleaned up using the same Amplification Kit.
Hybridization protocol
After the quality control assessment, 1.5 µg of cRNA from each experimental sample along with hybridization controls were hybridized for 16 hours to the MouseWG-6 v2.0 Expression BeadChips (Illumina, Inc., San Diego, CA) in the 58 °C Illumina Hybridization Oven. Washing, staining with streptavidin-Cy3 (GE Healthcare Bio-Sciences, Piscataway, NJ), and scanning were performed according to the Illumina Whole-Genome Gene Expression Direct Hybridization Assay Guide (revision A). Two BeadChips were used in this study, each containing six arrays.
Scan protocol
The arrays were scanned using an Illumina BeadArray Reader. Each array image was visually screened to discount for signal artifacts, scratches or debris.
Description
replication 2
Data processing
The raw bead-level files were processed with Illumina® BeadStudio 3.1.3, Gene Expression Module v3.4.0 (Illumina), without background correction or normalization, to get one value per beadtype for each array. These beadtype values were normalized and analyzed in R (R Development Core Team) using the beadarray package (Dunning et al., 2007) from the Bioconductor Project (Gentleman et al., 2004). Background fluorescence was corrected by subtracting the mean of the negative control beadtypes; negative and zero values after background correction were set to 0.5. Quantile normalization was performed and data were log2 transformed. Differential expression was assessed using a linear model using the limma package (Smyth, 2005), which uses an empirical Bayes correction (Smyth, 2004) that helps to improve power by borrowing information across beadtypes. BeadStudio’s Detection p-values were used to filter out 21,572 beadtypes that were not detected (p > 0.05) above the negative controls in any of the 12 samples. The remaining 23,709 beadtypes were input to Ingenuity Pathways Analysis as the reference set.
Submission date
Jul 23, 2010
Last update date
Jul 23, 2010
Contact name
Diana Cheng
Organization name
University of Illinois Urbana Champaign
Department
Natural Resources and Environmental Sciences
Lab
Mary Ann Lila Lab
Street address
1115 Plant Sciences Laboratory, 1201 South Dorner Drive
