Abstract
Epigenetics is believed to provide great chances for a better understanding of the development and treatment of many diseases where the analysis of genomic DNA has so far failed to provide conclusive answers. Methylcytosine is a frequently used quantitative marker of epigenetic studies. Since immediate analysis of sampled material is in most cases not possible, storage time and conditions are critical aspects regarding the quality of genomic DNA and reliability of analysis. Blood is frequently used for such analyses. We, therefore, collected blood samples of ten volunteers and stored them under various conditions for ten months: -70°C, -20°C, 2–8°C and room temperature. An additional aliquot was frozen at -70°C and thawed once a week at room temperature. We then compared the DNA extraction yields and methylation status in relation to storage time and conditions. We found significantly lower DNA extraction yields (up to -97.45%; p ≤ 0.001) as well as significantly higher methylation levels after ten months of storage (up to +42.0%; p ≤ 0.001). These results suggest that storage time has an important influence on DNA analyses of blood samples for all storage conditions. This might be due to differences in stability of methylated and non-methylated DNA. Our study indicates that storage conditions and time may be a critical factor for epigenetic methylation studies and require rigorous validation. For reliable analyses we, therefore, recommend to perform epigenetic analysis directly after sample collection.
Highlights
Epigenetic studies have become widespread and are continually gaining in importance
The yield of genomic DNA (gDNA) after the extraction decreased in line with storage time (Fig 1)
The cell lysis step of the gDNA extraction protocol had to be repeated in order to obtain an adequate amount of material
Summary
Epigenetic studies have become widespread and are continually gaining in importance. Researchers believe that epigenetics will in future provide the new insights into the development and the treatment of diseases which genetics was not able to explain so far [1, 2]. Methylcytosine is the key molecule in epigenetics and serves as a quantitative marker which changes throughout lifetime [3]. Stable sample material is essential for the reliability of analytical results of epigenetic studies. The most frequently used sample material is human whole blood, from which genomic DNA (gDNA) is extracted. Immediate gDNA extraction and analysis is often not viable.
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