Absolute rna miniprep kit

Fibroblasts were cultured on 60 mm diameter Petri dishes. Once the cells reached 95% confluence, they were treated with secosteroids (100 nM final concentration) diluted in media containing stripped serum, one plate per treatment, per experiment. Cells were harvested 24 h later for RNA isolation and supernatant was collected for collagen assays. Total RNA was isolated using an Absolute RNA miniprep kit (Agilent Technologies, Santa Clara, CA, USA), and quantified using a NanoDrop-2000 (Biotek, NJ, USA) spectrophotometer. cDNA was synthesized using a cDNA synthesis kit (Transcriptor First Strand cDNA Synthesis Kit, Roche, Indianapolis, IN, USA). PCR was performed in triplicates with Luminaris HiGreen low ROX PCR reagent (ThermoFisher, Waltham, MA, USA), as previously described. Samples were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or Cyclophilin B mRNA expression levels. Data for fibroblasts treated with vitamin D3 hydroxyderivatives were calculated as ΔΔCT normalized to an endogenous reference and analyzed using GraphPad Prism 9 statistical software and the t-test. Data are presented as the fold-change in gene expression in comparison to the vehicle (EtOH) in graphical form. Student’s t-test was used for statistical analysis (* p < 0.5; ** p < 0.1). Table 1 lists the primer sequences used for PCR.

RNA extraction from FACS-purified HFSCs was done using the absolute RNA miniprep kit as described in the manufacturer’s procedure (Agilent Technologies). The RNA quality was assessed by the Agilent RNA 6000 Pico kit on the Agilent 2100 bioanalyzer. For microarray analysis, 1 ng RNA was amplified by using the Gene Chip® WT Pico amplification Kit (Affymetrix, USA) as per the manufacturer’s instructions. Differentially expressed genes were analysed using Transcriptome Analysis Console (TAC) software.