Btained by chromatin immunoprecipitation Title Loaded From File combined with high throughput sequencing (F the ADP-linked ribose with mannose significantly decreases the activity, which ChIP-seq). These studies reported between 1,600 and 2,800 genomic VDR binding sites that locate both up- and downstream of the transcription start sites of primary vitamin D target genes. However, the three ChIP-seqCD14 and THBD as Transcriptomic Markers in VitDmetdatasets overlap only at some 200 genomic locations [25], i.e. there is a limited set of conserved targets of vitamin D and its receptor also on the genomic level. Sufficient exposure to natural UV-B radiation or adequate intake from diet or supplements is needed to achieve an optimal serum 25(OH)D3 concentration. However, the change in serum 25(OH)D3 concentrations can vary widely from person to person. Diet and sun exposure together 24195657 with age and adiposity in average account only for some 30 of the inter-individual variation in 25(OH)D3 serum concentrations [26]. Accordingly, genetic and epigenetic factors are responsible for the main variation in vitamin D concentrations [27?9]. Based on this wide inter-individual response variation, it is obvious that a “one-size-fits-all” approach will not work ideally for vitamin D supplementation. Therefore, we investigated in this study samples from 71 pre-diabetic participants of the VitDmet cohort, a 5-month high dose vitamin D3 intervention trial during Finnish winter [30], for their changes in serum 25(OH)D3 concentrations and the expression of primary vitamin D target genes in peripheral blood mononuclear cells and adipose tissue. Only the top half of a ranking concerning response to vitamin D provided a significant correlation between the changes of CD14 or THBD mRNA and serum 25(OH)D3 concentrations. We present CD14 and THBD as transcriptomic biomarkers, from which straightforward conclusions on the benefits of a vitamin D3 supplementation can be obtained.(10 mg/ml lidocaine without adrenalin). The adipose tissue samples were washed twice with phosphate-buffered saline, frozen quickly in liquid nitrogen and stored at 280uC until used for RNA extraction.RNA extraction and cDNA synthesisTotal RNA from PBMCs and adipose tissue samples was extracted using the TRIzol method followed by further purification with miRNeasy Mini Kit columns according to the instructions provided by the manufacturer (Qiagen). The RNA concentration and the A260/A280 ratio were 23727046 measured using the NanoDrop spectrophotometer, an acceptable ratio being 1.9?.1 [33]. The total RNA was reverse transcribed into cDNA using the High-Capacity cDNA Archive Kit (Applied Biosystems).Quantitative PCR (qPCR)qPCR reactions were performed using 250 nM of reverse and forward primers, 2 ml cDNA template (25 ng RNA/ml in the cDNA synthesis reaction and then diluted 1/10 prior to the PCR reaction) and the Roche LightCycler 480 SYBRGreen I Master (Roche) in a total volume of 10 ml. In the PCR reaction the hotstart Taq polymerase was activated for 10 min at 95uC, followed by 42 amplification cycles of 20 s denaturation at 95uC, 15 s annealing at primer-specific temperatures (Table S1 in File S1) and 15 s elongation at 72uC and a final elongation for 10 min at 72uC. PCR product specificity was monitored using post-PCR melt curve analysis. Relative expression levels were determined with the comparative delta threshold cycle (delta-Ct) method. Relative expression levels of the target genes were normalized to the internal reference genes B2M, GAPDH and HPRT1 (PBMCs) or GAPDH and RPLP0 (adipose tissue.Btained by chromatin immunoprecipitation combined with high throughput sequencing (ChIP-seq). These studies reported between 1,600 and 2,800 genomic VDR binding sites that locate both up- and downstream of the transcription start sites of primary vitamin D target genes. However, the three ChIP-seqCD14 and THBD as Transcriptomic Markers in VitDmetdatasets overlap only at some 200 genomic locations [25], i.e. there is a limited set of conserved targets of vitamin D and its receptor also on the genomic level. Sufficient exposure to natural UV-B radiation or adequate intake from diet or supplements is needed to achieve an optimal serum 25(OH)D3 concentration. However, the change in serum 25(OH)D3 concentrations can vary widely from person to person. Diet and sun exposure together 24195657 with age and adiposity in average account only for some 30 of the inter-individual variation in 25(OH)D3 serum concentrations [26]. Accordingly, genetic and epigenetic factors are responsible for the main variation in vitamin D concentrations [27?9]. Based on this wide inter-individual response variation, it is obvious that a “one-size-fits-all” approach will not work ideally for vitamin D supplementation. Therefore, we investigated in this study samples from 71 pre-diabetic participants of the VitDmet cohort, a 5-month high dose vitamin D3 intervention trial during Finnish winter [30], for their changes in serum 25(OH)D3 concentrations and the expression of primary vitamin D target genes in peripheral blood mononuclear cells and adipose tissue. Only the top half of a ranking concerning response to vitamin D provided a significant correlation between the changes of CD14 or THBD mRNA and serum 25(OH)D3 concentrations. We present CD14 and THBD as transcriptomic biomarkers, from which straightforward conclusions on the benefits of a vitamin D3 supplementation can be obtained.(10 mg/ml lidocaine without adrenalin). The adipose tissue samples were washed twice with phosphate-buffered saline, frozen quickly in liquid nitrogen and stored at 280uC until used for RNA extraction.RNA extraction and cDNA synthesisTotal RNA from PBMCs and adipose tissue samples was extracted using the TRIzol method followed by further purification with miRNeasy Mini Kit columns according to the instructions provided by the manufacturer (Qiagen). The RNA concentration and the A260/A280 ratio were 23727046 measured using the NanoDrop spectrophotometer, an acceptable ratio being 1.9?.1 [33]. The total RNA was reverse transcribed into cDNA using the High-Capacity cDNA Archive Kit (Applied Biosystems).Quantitative PCR (qPCR)qPCR reactions were performed using 250 nM of reverse and forward primers, 2 ml cDNA template (25 ng RNA/ml in the cDNA synthesis reaction and then diluted 1/10 prior to the PCR reaction) and the Roche LightCycler 480 SYBRGreen I Master (Roche) in a total volume of 10 ml. In the PCR reaction the hotstart Taq polymerase was activated for 10 min at 95uC, followed by 42 amplification cycles of 20 s denaturation at 95uC, 15 s annealing at primer-specific temperatures (Table S1 in File S1) and 15 s elongation at 72uC and a final elongation for 10 min at 72uC. PCR product specificity was monitored using post-PCR melt curve analysis. Relative expression levels were determined with the comparative delta threshold cycle (delta-Ct) method. Relative expression levels of the target genes were normalized to the internal reference genes B2M, GAPDH and HPRT1 (PBMCs) or GAPDH and RPLP0 (adipose tissue.
