Puregene core kit b

Total RNA was prepared from mouse tissues using Trizol® Reagent (Invitrogen, #15596-026). RNA concentration was determined with a NanoDrop spectrophotometer (Thermo Scientific, NanoDrop™ 2000c) and quality was assessed by agarose gel electrophoresis. cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen, 18080-051). qRT-PCR was performed with SYBR Green probes using the Applied Biosystems 7900HT Fast Real-Time PCR system. Results were expressed as the fold-change in transcript levels.
For mitochondrial DNA copy quantification, total DNA were isolated from adipose tissue by using Puregene Core Kit B (Qiagen) following the manufacture’s instruction. Relative amounts of nuclear DNA and mtDNA were determined by qRT-PCR. We used NADH dehydrogenase flavoprotein 1-coding gene for quantification of nuclear DNA and mitochondrial cytochrome c oxidase 2-coding gene, mitochondrial D-loop for mitochondrial DNA quantification.

DNA was isolated using the Puregene Core Kit B (Qiagen). 1μL of molecular grade glycogen (Thermo Scientific) was added to each sample and DNA was allowed to precipitate overnight at −20°C followed by resuspension in water. DNA methylation analysis of the CD16 loci was carried out using the MassARRAY EpiTYPER assay (Agena Biosciences) (29 (link)). In short, genomic DNA was subjected to bisulfite treatment using the EZ DNA methylation kit (Zymo Research). Regions of interest were amplified with PCR primers specific for the FCGR3A or FCGR3B gene loci (primers listed in Supplementary Table 1) using the hg19 genome assembly from the UCSC genome browser (https://genome.ucsc.edu). PCR products were in vitro transcribed and fragmented with RNase A (Agena) to generate oligonucleotides that were subsequently analyzed via Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-ToF) mass spectrometry. Ratios of unmethylated versus methylated oligo signals were utilized to calculate the percentage of DNA methylation for individual CpG dinucleotides. To examine the specificity of FCGR3A and FCGR3B assays, amplified sequences included non-CpG sequence variants that produced quantifiable differences in the mass spectra. Primers used for the final analysis were determined to be highly specific.