normalization data transformation protocol
Data was processed using Bioconductor Software with VST transformation and RSN normalization. ID_REF = VALUE = Log2 RSN normalized
array scanning protocol
Arrays were scanned with an iScan reader, and data were extracted and analyzed with BeadStudio Software (Illumina).
Amplified and labeled cRNA was quantified (Ribogreen assay) and hybridized to Mouse Ref-8 version 2.0 BeadChip Arrays (Illumina) overnight at 58°C. Following hybridization, arrays were washed, blocked, stained, and dried (Little Dipper Processor).
RNA was then amplified and labeled (Ambion TotalPrep Kit), and cDNA was synthesized using robotic-assisted magnetic capture. Using the cDNA template, biotinylated cRNA was produced via an in vitro transcription reaction.
nucleic acid extraction protocol
Skeletal muscle RNA was extracted using TRIzol solution (Invitrogen) and then purified using the RNeasy kit and RNase Free DNase Set (Qiagen).
sample treatment protocol
Quadriceps femoris muscles were harvested from 22-month-old muscle-specfic ATF4 knockout (ATF4 mKO) mice and littermate controls. mRNA levels in ATF4 mKO muscles were normalized to levels in littermate control muscles.