Dneasy blood tissue dna extraction kit

After synchronization by serum starvation for 24 hours, chondrocytes and osteoblasts were treated with AA (50–100 μg/mL; 284–568 μM) for 3 days. DNA was extracted using the DNEasy Blood & Tissue DNA extraction kit (Qiagen, Germantown, MD, USA). 5-hmC was measured by dot blot as follows. Briefly, DNA was deposited on nitrocellulose membranes in 1 μL droplets containing a series of 2-fold dilutions beginning at 100 ng/μL, UV crosslinked to the membrane (120,000 μJ/cm²), dried at 80°C, rinsed with 2X saline-sodium citrate, and probed with a 1:1000 dilution of 5-hmC antibody (Diagenode, Denville, NJ, USA) for 1 hour before chemiluminescent imaging. Blots were quantitated using ImageJ. 5-hmC ELISA was performed using a kit (Epigentek, Farmingdale, NY, USA) according to manufacturer’s instructions.

Total DNA and RNA were extracted simultaneously using the AllPrep DNA/RNA mini extraction kit (Qiagen, Crawley, UK), or separately using the GenElute Mammalian Total RNA miniprep kit (Sigma, Dorset, UK) or the DNeasy Blood & Tissue DNA extraction kit (Qiagen, Crawley, UK). Data presented in this manuscript utilise DNA extracted using both the DNeasy and AllPrep kits, but despite a slightly reduced DNA yield using the AllPrep kit, the quality of extracted DNA was the same for both kits. RNA was quantified using the Agilent 2100 BioAnalyzer (Agilent Technologies UK Limited) and 150 ng reverse transcribed using the First Strand cDNA Synthesis Kit (Life Technologies, Paisley, UK). Messenger RNA for the TFAM, COX1 and Ins1 genes were detected using TaqMan hydrolysis probes obtained from Applied Biosystems (Life Technologies, Paisley, UK), and normalised to the reference gene β2-microglobulin (B2M). DNA was used to determine mtDNA copy number, as described below. Real-time PCR was conducted using the Roche LightCycler 480 thermo cycler (Roche Diagnostics Ltd) and PCR products were quantified fluorometrically using the LightCycler 480 Master I (Roche, Welwyn Garden City, UK) kit and TaqMan probes for RNA or the LightCycler 480 SYBR Green I Master (Roche, Welwyn Garden City, UK) kit for DNA. Quantification of gene expression was performed using the Delta Ct (ΔCt) method [28] (link).

We used 24 tissue samples (Skeletal muscle) from sables, of which, 12 were from the areas (Genhe and Tahe) near the Greater Khingan Mountains, others were from the areas (Shangzhi and Mudanjiang) between the Lesser Khingan Mountains and the Changbai Mountains (Figure 1). Total genomic DNA of M. zibellina was extracted from tissue samples using the DNeasy Blood & Tissue DNA Extraction Kit (Qiagen) following the protocol of the manufacturer.