DNA methylation is widely recognized as a major player regulating normal gene expression and facilitating chromatin organization within cells. Research into abnormal DNA methylation that leads to human diseases like cancer could therefore provide valuable insights for disease development diagnosis and potential therapies.
DNA is made up of 4 nucleotides – cytosine, guanine, thymine and adenine. Methylated DNA includes a methyl (CH3) group added to the DNA strand itself, often to the fifth carbon atom of a cytosine ring. In humans, 5-methyl cytosine (5-mC) is found in approximately 1.5% of gDNA and occurs exclusively in the context of paired symmetrical methylation of a CpG site. CpG sites are regions of DNA where a cytosine is followed by a guanine.
The most popular method used to analyze DNA methylation is bisulfite conversion. Bisulfite treatment of the target DNA results in the conversion of unmethylated cytosine into uracil, which appears as thiamine after PCR amplification. The methylated cytosine will remain unchanged, still appearing as cytosine after PCR amplification.
The 5 major challenges in bisulfite conversion are:
- – Preparation of the sample to enable efficient bisulfite conversion
- – Effective DNA protection during bisulfite conversion (conversion can cause DNA fragmentation)
- – Complete conversion of all unmethylated cytosines – this is required for correct results interpretation
- – High recovery and concentration of bisulfite converted DNA for sensitive analysis
- – Fast time to result, which increases throughput and efficiency
QIAGEN has developed dedicated kits which not only can be used for direct bisulfite conversion of DNA from blood, cells, tissue samples or FFPE slices, but also provide solutions to overcome the different challenges mentioned above. The kits overcome these challenges by:
- – Providing a unique DNA-protecting buffer
- – Confirming correct pH and mixing with a pH indicator
- – Providing a ready-to-use bisulfite solution which not only reduces the actual bisulfite conversion time to as little as 30 minutes, but also improves the shelf life up to 6 months
- – Providing highly concentrated bisulfite DNA (bsDNA) in as little as 10 µl elution buffer
Overview of QIAGEN solutions for reliable bisulfite conversion:
|Product||Key Advantages||New Features|
|EpiTect Fast DNA Bisulfite Kit||Highly concentrated DNA suitable for all downstream applications; enhanced sensitivity of downstream applications due to minimal DNA fragmentation.||Low elution volume, ready-to-use bisulfite solution and fast conversion time combined with proven DNA protection.|
|EpiTect Fast LyseAll Bisulfite Kit||Minimized loss of sample material due to integrated sample lysis; high DNA concentration enables use in all downstream applications.||Integrated sample lysis (cells, tissue, blood) ready-to-use bisulfite solution and fast conversion time and low elution volume combined with proven DNA protection.|
|EpiTect Fast FFPE Bisulfite Kit||Highest yield, maximized conversion and highest concentration of converted DNA from as little as one FFPE slice.||Optimized DNA solution, ready-to-use bisulfite solution and fast conversion time and low elution volume combined with proven DNA protection.|
In addition it is recommended to build in a quality control step in order to quantify bsDNA using a spectrophotometer like the QIAxpert to eliminate bad samples from the workflow. Be aware that bisulfite converted DNA resembles RNA more than DNA which means that you need to use a value of 40 µg/mL for A260 = 1 and that the A260/A280 ratio of the bisulfite converted DNA should be between 1.7 and 1.9.
Want to know more about DNA methylation analysis?