Mice were allowed ad libitum access to food or fasted for 24 h before RNA was harvested from the tibilais anterior muscle.
Growth protocol
Not applicable
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TRIzol solution (Invitrogen) according to the manufacturer’s instructions.
Label
biotin
Label protocol
50 ng total RNA was converted to SPIA amplified cDNA using the WT-Ovation Pico RNA Amplification System, v1 (NuGEN Technologies, San Carlos, CA, Cat. #3300) according to the manufacturer’s recommended protocol. The amplified SPIA cDNA product was purified through a QIAGEN MinElute Reaction Cleanup column (QIAGEN Cat #28204) according to modifications from NuGEN. Four μg of SPIA amplified DNA were used to generate ST-cDNA using the WT-Ovation Exon Module v1 (NuGEN Technologies, Cat #2000) and again cleaned up with the Qiagen column as above. Five μg of this product were fragmented (average fragment size = 85 bases) and biotin labeled using the NuGEN FL-Ovation cDNA Biotin Module, v2 (NuGEN Technologies, Cat. #4200) per the manufacturer’s recommended protocol.
Hybridization protocol
Biotin-labeled cDNA was mixed with Affymetrix eukaryotic hybridization buffer (Affymetrix, Inc., Santa Clara, CA), placed onto Mouse Exon 1.0 ST arrays, and incubated at 45º C for 18 h with 60 rpm rotation in an Affymetrix Model 640 Genechip Hybridization Oven.
Scan protocol
Following hybridization, the arrays were washed, stained with streptavidin-phycoerythrin (Molecular Probes, Inc., Eugene, OR), signal amplified with antistreptavidin antibody (Vector Laboratories, Inc., Burlingame, CA) using the Affymetrix Model 450 Fluidics Station. Arrays were scanned with the Affymetrix Model 3000 scanner with 7G upgrade and data were collected using the using the GeneChip operating software (GCOS) v1.4.
