Methylflash hydroxymethylated dna quantification kit

Nuclei were extracted from rat hippocampi using an EpiQuik™ nuclear extraction kit (Epigentek, Brooklyn, New York, USA). The global 5-mC and 5-hmC in DNA were quantified using the MethylFlash Methylated DNA Quantification Kit (Cat #P-1034, Epigentek) and the MethylFlash Hydroxymethylated DNA Quantification Kit (Cat #P-1036, Epigentek) according to the manufacturer’s instructions.

Genomic DNA of liver tissues from all groups was extracted using QIAamp DNA Kits (QIAGEN, Germantown, MD, US) according to the manufacturer’s protocols. The level of epigenetic changes was determined by analysis of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) levels in the liver using a MethylFlash Methylated DNA Quantification Kit and a MethylFlash Hydroxymethylated DNA Quantification Kit (Epigentek, Brooklyn, NY, United States).

The isolation of genomic DNA was conducted using the FitAmp^TM Blood and Cultured Cell DNA Extraction Kit (EpiGentek, Farmingdale, NY, USA) according to the manufacturer’s instructions. Following this, the Methylflash Methylated DNA Quantification Kit (Epigentek, Farmingdale, NY, USA) and the MethylFlash HydroxyMethylated DNA Quantification Kit were used in order to detect methylated and hydroxymethylated DNA. In brief, these kits are based on the specific antibody detection of 5-mC and 5-hmC residues, which trigger an ELISA-like reaction that allows colorimetric quantification of global DNA methylation and 5-hydroxymethylation at 450 nm. Both kits were used following the manufacturer’s instructions, including internal standards for methylated and hydroxymethylated DNA.

Isolation of genomic DNA from 12 samples (n = 4 per group) was conducted using the FitAmpTM Blood and Cultured Cell DNA Extraction Kit according to the manufacturer’s instructions. Then, Methylflash Methylated DNA Quantification Kit (Epigentek, Farmingdale, NY, USA) and MethylFlash HydroxyMethylated DNA Quantification Kit were used in order to detect methylated and hydroxymethylated DNA. Briefly, these kits are based on specific antibody detection of 5-mC and 5-hmC residues, which trigger an ELISA-like reaction that allows colorimetric quantification by reading absorbance at 450 nm using a Microplate Photometer.

To independently assess quantitative cellular methylation, 25μm coronal sections were obtained from flash-frozen cerebellum at ages P8 (N = 8) and P29 (N = 8) using a Leica Cryostat CM1900. Sections were mounted onto 2μm thick PEN membrane slides (Microdissect Gmbh, Herborn, Germany) and stained with 4X Thionin solutions. Slides were further dehydrated in series 50%, 75%, 90%, and 100% ethanol. Sections were viewed under 10X magnification using a Leica laser microdissection microscope (Leica CTR6500). The cerebellar layers EGL, IGL and PCL were morphologically discernible by Thionin stain and dissected by laser into a flat cap 0.65mL Eppendorf tube containing 65μL of DNA extraction buffer (Arcturus Picopure DNA Extraction kit, Applied Biosciences). DNA was processed according to manufacturer’s instructions. DNA was next purified using DNA Clean and Concentrator (Zymo, Irving, CA) according to manufacturer’s instructions.
100ng and 200ng of purified cellular gDNA was used for MethylFlash^™ Methylated DNA Quantification Kit and MethylFlash^™ Hydroxymethylated DNA Quantification Kit respectively, according to manufacturer’s instruction (Epigentek, Farmingdale, NY). Absolute quantification was performed using a five-point standard curve and further transformed to represent the percentage of methylated DNA (5mC or 5hmC) in the total DNA.