BENCHMARKS
Figure 2. Assessment of DNA fragmentation in the DNA samples extracted from fresh (-) and old (+) blood of three individuals: A, B and C. DNA was run on a 1% aga- rose gel. There is evidence of increased fragmentation in samples extracted from old blood stored at room temperature for 3 days. Fur- thermore, HpaII/MspI ratios determined using LUMA were higher in samples ex- tracted from old blood, es- pecially in sample A. Finally, HPLC was used to calibrate the dC/(dC+dmC)
ratios,
and these were found to be unchanged in old blood.
the Supplementary Material for details of the calculation.) Our results suggest that our modification corrects for the effects of DNA fragmentation on global methylation levels as determined by LUMA. However, for extremely sheared samples, the correction was not always sufficient to compensate for the effects of fragmentation, resulting in negative ratios (Figure 1A, sample 3; Table 2). Our data provide support for the
following conclusions: (i) Global DNA methylation levels determined by LUMA seem to be dependent on the integrity of the DNA samples, such that the more fragmented the DNA, the greater the error in estimation of the true methyl- ation level. (ii) Incorporation of signal intensities from undigested DNA into the HpaII/MspI ratio calculation results in more accurate methylation levels. We believe that this modification needs further improvement, though it is sufficient to suggest that methylation levels measured by LUMA are affected by the integrity of the DNA samples. Therefore, researchers using LUMA for global methylation detection should be
cautious about the integrity of their DNA samples, and we recommend utilizing this modification as a quality check for every sample to be analyzed with LUMA. Since global methylation levels may be useful indicators for various disorders and have been suggested as biomarkers for diseases such as cancer, it is important to consider any factor that might impact the accuracy of LUMA results.
Author contributions
E.H. was involved in the conception of the study. E.H., E.A.D., and J.J.D. developed the study. E.A.D. and S.K. executed the experiments. E.H. and E.A.D. analyzed the results. E.A.D. wrote the manuscript, and E.H., E.A.D., and S.K. edited the manuscript.
Acknowledgments
We thank Nathaniel Heintz from Rocke- feller University for his contribution to the HPLC analysis. Samples were processed in the Genomics Core Facility
at Stony Brook University Medical Center.
Competing interests The authors declare no competing interests.
References
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Received 15 January 2015; accepted 26 February 2015.
Address correspondence to Elif Aysimi Duman, Bogazici University South Campus, Department of Psychology, Istanbul, Turkey. E-mail: elif.duman@
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