Quest 5 hmctm dna elisa kit

The 5hmC content of genomic WBC DNA in patients with different prostate pathologies and control samples was determined by colorimetric ELISA with the Quest 5-hmC^TM DNA ELISA Kit (Zymo Research, Irvine, CA, USA; Cat No. D5426). This is the most cited ELISA-based global 5hmC quantification kit in the literature (over 60 citations according to Google Scholar), and provides scientists with a quick, cost-effective, and reliable alternative to chromatographic methods (e.g., LC-MS/MS, HPLC) that are used for this purpose, particularly for serial measurements, which was the case of our study. Assays were conducted in duplicate per each sample, according to the manufacturer’s instructions by loading 100 ng of DNA per well. To limit variation in measurements related to potential batch or procedural discrepancies, we have randomly rerun ELISA analysis for about a quarter of DNA samples. Optical density (OD) was red at 450 nanometers (nm) using a microplate Reader Stat Fax 4200 (Awareness Technology, Palm City, FL, USA). For absolute 5hmC quantification, a standard curve was obtained by plotting the various concentrations of the positive controls against the corresponding ODs.

Genomic DNA was isolated from PBMCs using Blood Genomic DNA Purification Kit (GMbiolab Co., Ltd., Taiwan, China) and quantified by Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Genomic DNA methylation and hydroxymethylation were determined by measuring 5-mC and 5-hmC using 5-mC DNA ELISA Kit and Quest 5-hmC^TM DNA ELISA Kit (Zymo Research, Irvine, CA, USA), respectively [46 (link), 47 (link)]. These are the most cited ELISA-based global 5-mC and 5-hmC quantification kit in the literature, and provides scientists with a quick, cost-effective and reliable alternative to chromatographic methods [48 (link), 49 (link)]. Assays were performed according to the manufacturer’s instructions loading 100 ng of DNA per well. The absorbance at 405 nm was measured using a Multiskan Spectrum (Thermo Electron Corporation, Waltham, MA, USA).

The 5-hydroxymethylcytosine of genomic DNA was detected by using Quest 5-hmC TM DNA ELISA Kit (ZYMO Research Corp. Irvine, USA). StromaNKtert cells were collected and genomic DNA was extracted with Tissue & Cell Genomic DNA purification kit (GMbiolab Co. Ltd, Taiwan). Genomic DNA was denatured at 98 °C for 5 min and single-stranded DNA was diluted to final concentration 1 ng/μL. Diluted DNA (100 μL) was added into each well coated with anti-5-hydroxymethylcytosine polyclonal antibody and incubated at 37 °C for 1 h. After incubation, the plates were washed 3 times and incubated with horseradish peroxidase (HRP)-conjugated anti-DNA antibody at 37 °C for 30 min. Developing reagent was added and allowed for color development at room temperature for 10 min. Finally, the absorbance was measured at 450 nm with ELISA plate reader.