Measurement of 5mC and 5hmC

The methods for DNA isolation from the mouse BM cells and tissue lysates, using the DNeasy kit (Qiagen, ValenciaCalifornia), have been previously presented.^40,49 Prior to using for the measurements of levels of global 5mC and 5hmC, DNA concentrations and purities were measured using the BioPhotometer (Eppendorf Co). Subsequently, we used commercially available ELISA kits for the detection of global 5mC and 5hmC (Zymo Research, Inc, Irvine, California) to measure the percentage of global 5mC and 5hmC in the DNA samples isolated from the BM cells and the lung/testis lysates of these mice. The methods suggested by the manufacturer were followed. In brief, the levels of global 5mC and 5hmC were measured using a microplate spectrophotometer (Molecular Devices) at 405 nm. Thereafter, the % 5mC and % 5hmC were calculated from a standard curve obtained using the control DNA set provided by the manufacturer. The measurements of 5mC and 5hmC in the DNA sample from each tissue of each mouse were also done in duplicate (using 200 ng of DNA per well). Finally, the median, mean, and SE values of global 5mC and global 5hmC were obtained for each treatment group.