Local aneasthesia with 2% (w/v) lidocaine, incision of the skin and fascia, ~100 mg mucle tissue needle biopsy ~15 cm above the knee according to the Bergström technique, immediately frozen in liquid nitrogen and stored at -80°C
Growth protocol
not applicable
Extracted molecule
total RNA
Extraction protocol
Frozen muscle biopsy samples were ground using a mortar and pestle, placed in RNA-Bee, which contains phenol and guanidine thiocyanate (Tel-test, Friendswood, TX, USA), and homogenised using an T25 ultra-turrax dispenser (IKA, Staufen, Germany). RNA was isolated using phenol/chloroform extraction, and purified according to the RNeasy mini kit clean-up protocol including an on-column DNase digestion (Qiagen, Hilden, Germany). The RNA concentration and purity were measured with a Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA) and RNA integrity was analysed on an Agilent Bioanalyser (Santa Clara, CA, USA).
Label
biotin
Label protocol
According to the Affymetrix GeneChip whole transcript sense target labelling assay
Hybridization protocol
According to the Affymetrix GeneChip whole transcript sense target labelling assay
Scan protocol
According to the Affymetrix GeneChip Expression Wash, Stain and Scan User Manual
Data processing
The Affymetrix CEL files were imported into Genomic Suite software (Version 6.4, Partek, St. Louis, MO, USA). Probe sets were normalised using the Robust Multiarray Analysis (RMA) algorithm including background correction for probe GC content. Transcript summaries were calculated using the mean intensities of the corresponding probe sets, representing the quantitative gene expression levels.
