Quantigene plex 2

SOCS3, Myf5, and interleukin 6 (IL-6) mRNA levels were analyzed in samples from the first experiment, by using the QuantiGene Plex 2.0 assay (Panomics/Affymetrix, Fremont, CA, USA) according to the manufacturer's instructions. Briefly, muscles were homogenized in Affymetrix proprietary homogenization buffer with proteinase K (1:60, muscle weight to volume), incubated at 65°C for 30 min, and centrifuged at 12,000 rpm for 20 min at 4°C, and the supernatant was collected for subsequent RNA quantification. Lysates where transferred to a 96-well capture plate with target-specific probe sets, and the plate was sealed and incubated overnight at 55°C for hybridization. Next, the signals of the selectively captured target RNAs were amplified via sequential wash and hybridization steps, followed by the addition of the streptavidin-conjugated R-phycoerythrin, which emits a fluorescent signal that is proportional to the amount of target mRNA present in the sample. mRNA concentrations were normalized to the geometric average of 4 internal control genes (HPRT-1, GUSB, PPIA, and HMBS) and expressed as median fluorescence intensity (MFI). Atrogin-1 and muscle RING-finger 1 (MuRF1) mRNA expression was measured in samples from experiment 4, with predesigned rat primer and probe sequences commercially available from Applied Biosystems (Foster City, CA, USA; ref. 14 (link)).