Cells (buffy coat), which were stored at -80 until further processing

Cells (buffy coat), which were stored at -80 until further processing [66, 67].DNA extraction and bisulfite treatmentDNA was isolated from the buffy coat using the QIAamp DNA Blood Mini Kit (Qiagen, Zebularine web Hilden, Germany) by following the manufacturer’s instructions. EpiTect Bisulfite Kit (Qiagen) was used for bisulfite conversion and cleanup of DNA, during which unmethylated cytosines were converted to uracils and the methylated cytosines were conserved [20, 68]. DNA quality and quantity were examined with a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT, USA) and then stored in aliquots at -20 until further assay.Measurement of mtDNAnThe methylation of D-loop region was determined by methylation-specific PCR as descried previously [20, 68]. Briefly, the D-loop sequence 16024?76 (1,122 bp) of the Homo sapiens mitochondrion genome (gi|251831106: c576-1, c16569-16024) was used to identify the CpG island (426?76) and design primers for PCR analysis. The following two pairs of primers were designed: one pair was specific for bisulfite-modified methylated DNA, and the other pair was specific for bisulfite-modified unmethylated DNA amplifying heavy strand. The primers used in this study were TAGGAATTAAAGATAGAT ATTGCGA (forward, starting position at 434 nt) and 5ACTCTCCA TACATTTAATATTTTCGTC-3 (reverse, starting position at 539 nt) for methylated D-loop; 5GGTAGGAATTAAA PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27362935 GATAGATATTGTGA-3 (forward, starting position at 432 nt) and 5-ACTCTCCATACATT TAATATTTTCATC-3 (reverse, starting position at 539 nt) for unmethylated D-loop. The bisulfite-modified DNA was used as a template for methylation-specific PCR (MSP) on a ViiATM 7 Real-Time PCR System, using SYBR?Green PCR Master Mix (Life Technology, Grand Island, NY, USA). Two MSPs were performed simultaneously to detect the methylated (amplicon size; 106 bp) and unmethylated (amplicon size; 108 bp) D-loop for each sample. The percentage of methylated DNA is calculated as described previously [20, 68].StatisticsThe data are expressed as the mean ?SE unless otherwise specified. Logarithm-transformed data were used for the analysis of skewed variables, such as HOMA-IR and mtDNAn. Pearson’s correlation and regression analysis was applied to evaluate the relationships among Actidione solubility mtDNAn and the metabolic indexes. Statistical significance was set at a probability level of p < 0.05.Additional filesMitochondrial DNA copy number (mtDNAn) was measured as previously described [10, 41]. Briefly, 40 ng total DNA was used for real-time PCR with the iQTM SYBR?Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) on a ViiATM 7 Real-Time PCR System (Life Technology, Grand Island, NY, USA). The primers used inAdditional file 1: Figure S1. Scatter plot of the measurements and demographic characteristics of lean (n = 8) and obese (n = 32) participants in this study. The middle lines indicate the mean values, and the other two shorter lines indicate SE *p < 0.05; **p < 0.001; ***p < 0.0001. Additional file 2: Figure S2. Age-matched analysis of mtDNAn in lean (n = 7) and obese (n = 8) participants. (A) No significant difference existed between the ages of lean (n = 7) and obese (n = 8) participants. (B)Zheng et al. Clinical Epigenetics (2015) 7:Page 8 ofComparison of mtDNAn between lean (n = 7) and obese (n = 8) participants. The data were presented as mean ?SE. *p < 0.05; NS, not significant. Additional file 3: Figure S3. Regression analyses of mtDNAn with metabolic parameters and demographic.Cells (buffy coat), which were stored at -80 until further processing [66, 67].DNA extraction and bisulfite treatmentDNA was isolated from the buffy coat using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) by following the manufacturer's instructions. EpiTect Bisulfite Kit (Qiagen) was used for bisulfite conversion and cleanup of DNA, during which unmethylated cytosines were converted to uracils and the methylated cytosines were conserved [20, 68]. DNA quality and quantity were examined with a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT, USA) and then stored in aliquots at -20 until further assay.Measurement of mtDNAnThe methylation of D-loop region was determined by methylation-specific PCR as descried previously [20, 68]. Briefly, the D-loop sequence 16024?76 (1,122 bp) of the Homo sapiens mitochondrion genome (gi|251831106: c576-1, c16569-16024) was used to identify the CpG island (426?76) and design primers for PCR analysis. The following two pairs of primers were designed: one pair was specific for bisulfite-modified methylated DNA, and the other pair was specific for bisulfite-modified unmethylated DNA amplifying heavy strand. The primers used in this study were TAGGAATTAAAGATAGAT ATTGCGA (forward, starting position at 434 nt) and 5ACTCTCCA TACATTTAATATTTTCGTC-3 (reverse, starting position at 539 nt) for methylated D-loop; 5GGTAGGAATTAAA PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27362935 GATAGATATTGTGA-3 (forward, starting position at 432 nt) and 5-ACTCTCCATACATT TAATATTTTCATC-3 (reverse, starting position at 539 nt) for unmethylated D-loop. The bisulfite-modified DNA was used as a template for methylation-specific PCR (MSP) on a ViiATM 7 Real-Time PCR System, using SYBR?Green PCR Master Mix (Life Technology, Grand Island, NY, USA). Two MSPs were performed simultaneously to detect the methylated (amplicon size; 106 bp) and unmethylated (amplicon size; 108 bp) D-loop for each sample. The percentage of methylated DNA is calculated as described previously [20, 68].StatisticsThe data are expressed as the mean ?SE unless otherwise specified. Logarithm-transformed data were used for the analysis of skewed variables, such as HOMA-IR and mtDNAn. Pearson’s correlation and regression analysis was applied to evaluate the relationships among mtDNAn and the metabolic indexes. Statistical significance was set at a probability level of p < 0.05.Additional filesMitochondrial DNA copy number (mtDNAn) was measured as previously described [10, 41]. Briefly, 40 ng total DNA was used for real-time PCR with the iQTM SYBR?Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) on a ViiATM 7 Real-Time PCR System (Life Technology, Grand Island, NY, USA). The primers used inAdditional file 1: Figure S1. Scatter plot of the measurements and demographic characteristics of lean (n = 8) and obese (n = 32) participants in this study. The middle lines indicate the mean values, and the other two shorter lines indicate SE *p < 0.05; **p < 0.001; ***p < 0.0001. Additional file 2: Figure S2. Age-matched analysis of mtDNAn in lean (n = 7) and obese (n = 8) participants. (A) No significant difference existed between the ages of lean (n = 7) and obese (n = 8) participants. (B)Zheng et al. Clinical Epigenetics (2015) 7:Page 8 ofComparison of mtDNAn between lean (n = 7) and obese (n = 8) participants. The data were presented as mean ?SE. *p < 0.05; NS, not significant. Additional file 3: Figure S3. Regression analyses of mtDNAn with metabolic parameters and demographic.