BioProject

Human skin fibroblasts from an individual with hereditary vitamin D-resistant rickets bearing a homozygous p. More...

Human skin fibroblasts from an individual with hereditary vitamin D-resistant rickets bearing a homozygous p.Arg30* VDR mutation [Damiani et al., Osteoporos Int 2015; 26(6):1819-23] and from an age/sex-matched control were obtained from 4-mm punch biopsies of the forearm skin, after institutional board approval and with informed consent. Skin explants were fragmented in 6-well tissue culture plates and covered in complete AmnioMAX™ C-100 medium until attachment to surface; after approximately 12 days fibroblasts grown out of explants covered well surfaces completely. Secondary fibroblast cultures were maintained in high glucose DMEM supplemented with 10% FBS and 1% P/S. Fibroblasts were used for experiments between passages five to fifteen. Global gene expression of CO and MUT fibroblasts in response to 1,25D or ethanol vehicle (Veh) was analysed using microarrays. Six independent biological replicates were performed for each experimental condition: CO Veh, CO 1,25D, MUT Veh and MUT 1,25D. Cells were grown in 6-well plates and treated with 10 nM 1,25D or ethanol (1 ul/ml of medium) for 24 hours before RNA extraction. Based on quality control of extracted RNA performed with the 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), four samples of each condition were chosen for microarray gene expression analysis, all with RNA integrity number above 8.00. Samples were processed according to manufacturer’s instructions, starting with 200 ng total RNA. Altogether, sixteen samples of labeled fragmented cDNA were hybridized to GeneChip Human Gene 2.0 ST Arrays (Affymetrix). Array data was analysed using Partek Genomics Suite, and based on quality control one array (MUT_Veh_4) was excluded. A Benjamini-Hochberg-corrected p-value cut-off of 0.05 was used for selecting significant differentially expressed genes. Overall design: Four biological replicates of control fibroblasts treated with vehicle (arrays CO_Veh 1 to 4), four biological replicates of control fibroblasts treated with 1,25D (arrays CO_1,25D 1 to 4), three biological replicates of mutant p.Arg30* VDR fibroblasts treated with vehicle (arrays MUT_Veh 1 to 3) and four biological replicates of mutant p.Arg30* VDR fibroblasts treated with 1,25D (arrays MUT_1,25D 1 to 4) Less...