Imprint® Methylated DNA Quantification

The Imprint® Methylated DNA Quantification technology provides a rapid and reliable method to measure global DNA methylation shifts. Compared to the conventional methods of analyzing hypo- and hyper- global methylation like reverse HPLC, MALDI-TOF-MS, TLC, etc. Sigma’s Imprint®. Methylated DNA Quantification Kit (MDQ1), allows researchers to spend less time in the lab, with less hazardous components and still get highly accurate results.

Benefits of the kit ELISA based format Procedure complete in 4 easy to follow steps (Fig 1) All reagents including control methylated DNA included in the kit. Quickly measures changes in DNA methylation Detection limit is 5ng of fully methylated DNA Input DNA can be as low as 10ng-200ng (Fig 2, Fig 3) Complete, flexible and fast Process complete in 3.5 hours 96 well format allows option of studying single sample or high-throughput studies Extended range of DNA sample source Can be used with cells, tissues, plasma and other body fluids

		MDQ1 Standard Cruve, Methylated Control
		ng DNA	A450	Error
		200	0.594	0.047
		100	0.39	0.03
		50	0.251	0.018
		25	0.144	0.009
		12.5	0.072	0.0112
		0	0	0

Calculations

To calculate relative methylation levels compared to the control DNA or DNA from a normal sample, use the following steps.

Direct Calculation
Percent methylation compared to the control DNA

1. Average all control, sample, and blank absorbance replicates.
2. Subtract the average blank value from the average control and sample values.
3. Divide the background subtracted average sample by the background subtracted average normal sample.
4. Multiply by 100.
5. Sample calculation:
   Average Blank Absorbance = (A450) = 0.090
   Average Absorbance for 60 ng control DNA = 0.785
   Average Absorbance for 60 ng sample DNA = 0.654
   
      0.654 - 0.090 x 100 = 81.15%
      0.785 - 0.090
   
   The sample DNA has a global methylation level that is 81.15% of the control DNA.

Calculation from a standard curve
Percent methylation relative to DNA used to generate the standard curve

1. Determine the concentration of DNA in the sample(s) by A260 or fluorimetric means.
2. Create a standard curve by plotting background subtracted A450 versus control DNA (see example)
3. For each background subtracted sample reading, calculate the % methylation as below:
   Methyl DNA (ng) = [A450 background subtracted – intercept] / slope
                                                             ng in sample

Note: in many or most cases the intercept will be zero.

View the Imprint® Methylated DNA Quantification Troubleshooting Questions

View the poster presented at ASHG, Nov 12-1, 2008 at Philadelphia "Method to Measure Global DNA Methylation"
 

Azacytadine, a known demethylating agent, was added directly to culture media. Cells were harvested after 48 hours. DNA was isolated using GenElute and global methylation was measured. 40 ng of DNA from treated and non-treated CHO cells was analyzed.

Methylation status of two lots of Sss I treated DNA was measured by LC-MS. Varying amounts of DNA was also analyzed via the MDQ1 kit. As demonstrated by the graph, the difference in methylation states can easily be detected and the signal generated is proportional to the amount of DNA used in the procedure.

Ordering information

Products to Support the Epigenetics Workflow using this kit

DNA extraction and modification

DNA-protein interaction & ChIP validated antibodies

Mission shRNA for functional genomic studies