Profiling of RNA expression in soleus muscles from muscle-specific Gna13 knockout mice

Gna13 transmit extracellular signals from cell surface G protein-coupled receptors to intracellular effector molecules. We have characterized the Gna13-dependent regulatory network in muscle through transcriptional profiling of the mouse soleus in Gna13 muscle-specific knockout (Gna13 MKO) mice or wild-type littermates with floxed genotype (WT). We crossed Ckmm-Cre mice (Jackson laboratory #006475) with Gna13 flox/flox mice, which was provided from Dr. Offermanns S (Max Planc Institute). At 18 weeks of age, Gna13 flox/flox (WT) or Ckmm-Cre; Gna13 flox/flox (Gna13 MKO) mice were fasted overnight and sacrificed. Immediately after sacrifice, soleus muscles were snap-frozen and stored until RNA extraction (n=3 each). For the quality control, RNA purity and integrity were evaluated by OD 260/280 ratio, and analyzed by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, USA). The Affymetrix Whole transcript Expression array process was executed according to the manufacturer's protocol (GeneChip Whole Transcript PLUS reagent Kit). cDNA was synthesized using the GeneChip WT (Whole Transcript) Amplification kit as described by the manufacturer. The sense cDNA was then fragmented and biotin-labeled with TdT (terminal deoxynucleotidyl transferase) using the GeneChip WT Terminal labeling kit. Approximately 5.5 μg of labeled DNA target was hybridized to the Affymetrix GeneChip Mouse 2.0 ST Array at 45°C for 16hour. Hybridized arrays were washed and stained on a GeneChip Fluidics Station 450 and scanned on a GCS3000 Scanner (Affymetrix). Raw data were extracted automatically in Affymetrix data extraction protocol using the software provided by Affymetrix GeneChip® Command Console® Software (AGCC). After importing CEL files, the data were summarized and normalized with robust multi-average (RMA) method implemented in Affymetrix® Expression Console™ Software (EC). We exported the result with gene level RMA analysis and perfomed the differentially expressed gene (DEG) analysis. The comparative analysis between test sample and control sample was carried out using LPE test and fold change in which the null hypothesis was that no difference exists among 2 groups. False discovery rate (FDR) was controlled by adjusting p value using Benjamini-Hochberg algorithm.