Subcutaneous saline or oligo injections were administered to adult mice biweekly for 4 weeks.
Extracted molecule
total RNA
Extraction protocol
Quadriceps muscles were homogenized with a Polytron homogenizer in Trizol reagent according to the manufacturer's recommendations.
Label
biotin
Label protocol
Samples for mRNA profiling studies were processed by Asuragen, Inc., according to the company’s standard operating procedures. The purity and quantity of total RNA samples were determined by absorbance readings at 260 and 280 nm using a NanoDrop ND-1000 UV spectrophotometer. The integrity of total RNA was qualified by Agilent Bioanalyzer 2100 capillary electrophoresis. Total RNA (100 ng per sample) was used for preparation of biotin-labeled targets (cRNA) using Illumina’s TotalPrep Kit (Ambion Inc., Austin, TX) and one round of amplification. The cRNA yields were quantified by UV spectrophotometry and the distribution of transcript sizes was assessed using the Agilent Bioanalyzer 2100 capillary electrophoresis system.
Hybridization protocol
Labeled cRNA was used to probe MouseRef-8 Expression BeadChips. Hybridization, washing, and scanning of the Illumina arrays were carried out according to the manufacturer’s instructions. Briefly, cRNA/hybridization buffer mixtures were incubated at 65°C for 5 minutes, followed by brief centrifugation to collect. The mixtures were added to BeadChips and then BeadChips inserted into hybridization chambers, and hybridization was carried out at 58°C for 16-18 hours at rocking speed set to 5.
Scan protocol
BeadChips were washed and stained in the following steps: 1) 55°C for 10 minutes static in HighTemp Wash Buffer in a Hybex waterbath, 2) ambient temperature shaking for 5 minutes in E1BC wash buffer, 3) ambient temperature shaking for 10 minutes in ethanol, 4) ambient temperature shaking for 2 minutes in E1BC buffer, 5) ambient temperature for 10 minutes rocking in Block E1 buffer, 6) ambient temperature for 10 minutes rocking in Block E1 buffer containing 1ug/ml Cy3-streptavidin, and 7) ambient temperature shaking for 5 minutes in E1BC buffer. BeadArrays were dried in a centrifuge for 4 minutes and scanned using an Illumina BeadArray Reader. Illumina BeadScan software was used to produce .idat, .xml, and .tif files for each array on a slide and .sdf files for each barcode on a slide of 8 arrays. Raw data were extracted using Illumina BeadStudio software v3. Following quality assessment, data from the replicate beads on each array were summarized into raw intensity values with and without background subtraction in an Excel report.
Description
5543266006_B
Data processing
Background subtracted data were quantile normalized with the bioconductor affy package. If the minimum value in a row was less than 2, a constant was added to all values in that row such that the minimum was increased to 2.
