Total RNA was extracted from quadriceps biopsies using the RNeasy fibrous tissue mini kit (Qiagen). RNA quality was determined by Bioanalyzer (Agilent), and samples with a RIN (RNA Integrity Number) <6 were excluded. RNA was prepared for whole transcriptome microarray analysis using the WT expression kit (Ambion).
Label
biotin*
Label protocol
For each sample, 200ng of total RNA was converted to cDNA using the Ambion Whole Transcript (WT) expression kit. The Affymetrix GeneChip WT Terminal Labelling Kit* was used to fragment and label 5.5µg of the prepared single-stranded cDNA.
Hybridization protocol
Samples were hybridised to Affymetrix HuGene 1.1 ST 16- or 24-PEG array plates for 16 hours using the GeneTitan platform (Affymetrix Ltd.).
Scan protocol
GeneChips were scanned using the GeneTitan platform (Affymetrix Ltd.).
Data processing
Raw signal intensity data were RMA-treated, summarizing at the level of Transcript Clusters (TC) using Affymetrix Power Tools (APT, version 1.16.1). Technical batch effects were identified via Principal Variance Component Analysis (PVCA 1.6.0), and the data adjusted accordingly using ComBat (SVA 3.12.0). TC not forming part of the main array design, and universally low expressed TC were removed.
