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Accurate DNA Methylation Analysis with Successful Bisulfite Conversion Webinar
The document outlines the importance of accurate DNA methylation analysis through successful bisulfite conversion, including the biological relevance of DNA methylation and the challenges of the conversion process. It highlights Qiagen's solutions, such as the EpiTect fast bisulfite kits, which offer streamlined procedures, reduced sample loss, and rapid results. The document emphasizes the benefits of these kits for efficient and reliable downstream analysis in various molecular biology applications.
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Accurate DNA Methylation Analysis with Successful Bisulfite Conversion Webinar
- 1. Sample to Insight AccurateDNA methylation analysis by successful bisulfite conversion Dr. Inga Irle, Global Product Manager
- 2. Sample to Insight LegalDisclaimer Accurate DNA methylation analysisby successful bisulfite conversion 2 QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of a disease. For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor.
- 3. Sample to Insight Agenda AccurateDNA methylation analysisby successful bisulfite conversion 3 Introduction to DNA methylation1 19 Analysis of DNA methylation2 20 Challenges of bisulfite conversion3 21 Overview of QIAGEN solutions for bisulfite conversion 4 22 Summary5 23
- 4. Sample to Insight Agenda AccurateDNA methylation analysisby successful bisulfite conversion 4 Introduction to DNA methylation1 19 Analysis of DNA methylation2 20 Challenges of bisulfite conversion3 21 Overview of QIAGEN solutions for bisulfite conversion 4 22 Summary5 23
- 5. Sample to Insight 1)Introduction to DNA Methylation DNA Methylation • Addition of a methyl (CH3) group at the 5- carbon of the cytosine ring • In humans, 5-mC is found in approximately 1.5% of gDNA • 5-mC occurs almost exclusively in the context of paired symmetrical methylation of a CpG site .CpG islands • In the bulk of genomic DNA, most CpG sites are heavily methylated • CpG islands remain unmethylated, thus allowing gene expression to occur • When a CpG island in the promoter region of a gene is methylated, expression of the gene is repressed Biological relevance • DNA methylation acts in concert with other epigenetic mechanisms to regulate normal gene expression and facilitate chromatin organization within cells. • Abnormal methylation has been associated with a variety of diseases, e.g. cancer • The analysis of of DNA methylation could provide value insights on disease development, diagnosis and therapy Accurate DNA methylation analysisby successful bisulfite conversion
- 6. Sample to Insight Agenda AccurateDNA methylation analysisby successful bisulfite conversion 6 Introduction to DNA methylation1 19 Analysis of DNA methylation2 20 Challenges of bisulfite conversion3 21 Overview of QIAGEN solutions for bisulfite conversion 4 22 Summary5 23
- 7. Sample to Insight 2)Analysis of DNA methylation - Principle of bisulfite conversion Accurate DNA methylation analysisby successful bisulfite conversion 7 Bisulfite conversion – the key step in DNA methylation analysis • Crucial step in methylation analysis workflow • Efficient base conversion required for accurate assessment of methylation status
- 8. Sample to Insight 2)Analysis of DNA methylation - workflow Accurate DNA methylation analysisby successful bisulfite conversion 8 EpiTect Fast Bisulfite Kits are compatible with various analysis methods DNA purification Bisulfite treatment Amplification Analysis • QIAamp • EpiTect Fast FFPE Bisulfite • EpiTect Fast LyseAll Bisulfite • QIAcube • QIAsymphony • EpiTect Fast DNA Bisulfite • QIAcube • EpiTect Whole Bisulfitome • EpiTect MSP • EpiTect MethyLight PCR • EpiTect HRM • EpiTect Control DNAs • PyroMark PCR • PyroMark CpG Assays • Rotor-Gene Q • Pyromark Q48 KitsInstruments
- 9. Sample to Insight Agenda AccurateDNA methylation analysisby successful bisulfite conversion 9 Introduction to DNA methylation1 19 Analysis of DNA methylation2 20 Challenges of bisulfite conversion3 21 Overview of QIAGEN solutions for bisulfite conversion 4 22 Summary5 23
- 10. Sample to Insight Challengesof bisulfite conversion Accurate DNA methylation analysisby successful bisulfite conversion 10 a) Sample preparation b) DNA protection during bisulfite conversion c) Complete conversionreaction d) Fast results e) High recovery and concentrationof bsDNAfor downstream analysis DNA purification Bisulfite treatment Amplification Analysis
- 11. Sample to Insight 3)Challenges of bisulfite conversion Accurate DNA methylation analysisby successful bisulfite conversion 11 a) Sample preparation • Samples are precious and not unlimited • Purity of the gDNA is very important for the success of complete bisulfite conversion, as protein contamination prevent efficient conversion • DNA purification is often required before bisulfite conversion, resulting in loss of DNA • Formalin-fixed paraffin embedded (FFPE) tissues are particularly challenging – because of the fixing process, it causes modifications to the DNA, including DNA-protein crosslinks
- 12. Sample to Insight 3)Challenges of bisulfite conversion Accurate DNA methylation analysisby successful bisulfite conversion 12 a) Sample preparation • EpiTect Fast Bisulfite Conversion Kits provide a very fast, streamlined procedure for efficient bisulfite conversion and purification of DNA directly from any starting material — blood, cells, tissue samples or FFPE slices • The kits function as an integrated system of solutions for direct bisulfite conversion of DNA from blood, cells, tissue samples or FFPE slices • Integrated lysis minimizes loss of sample material
- 13. Sample to Insight 3)Challenges during bisulfite conversion Accurate DNA methylation analysisby successful bisulfite conversion 13 b) DNA protection • Conversion requires ssDNA • Chemical conversion occurs at non-physiological pH and temperature which causes DNA fragmentation • The more break-points, the smaller the DNA fragments • The smaller the DNA, the higher the risk that downstream applications fail • Lower analysis sensitivity would need to be compensated by increasing amount of target DNA Bisulfite Conversion w/o DNA protection Detectable DNA: 1/15 DNA molecules Detectable DNA: 10/15 DNA molecules
- 14. Sample to Insight 3)Challenges during bisulfite conversion Accurate DNA methylation analysisby successful bisulfite conversion 14 b) DNA protection Unique DNA Protect Buffer: • Fewer break-points • Enhanced performance of downstream applications • High sensitivity - less DNA is needed • Minimal DNA degradation ensurs high yields of DNA that can be analyzed
- 15. Sample to Insight 3)Challenges of bisulfite conversion Accurate DNA methylation analysisby successful bisulfite conversion 15 c) Complete conversion • Incomplete conversion of unmethylated cytosines leads to false positive methylation results • Complete denaturation of the DNA is necessary for efficient bisulfite conversion • Optimal pH needed for complete conversion of cytosine to uracil Complete conversion requires single-stranded DNA, optimum pH and temperature, as well as a well-balanced ratio of DNA to bisulfite. • DNA Protect buffer facilitates highly effective DNA denaturation and results in the formation of single-stranded DNA that is necessary for complete cytosine conversion • pH indicator confirms correct pH and correct mixing
- 16. Sample to Insight 3)Challenges of bisulfite conversion Accurate DNA methylation analysisby successful bisulfite conversion 16 c) Complete conversion EpiTect Fast Bisulfite Conversion Kits contain an innovative Bisulfite Solution that enables ultrafast, highly efficient bisulfite conversion, even on difficult-to-analyze samples such as X-chromosome genomic DNA derived from a female sample, where one of the 2 X-chromosomes is highly methylated: 16
- 17. Sample to Insight 3)Challenges of bisulfite conversion Accurate DNA methylation analysisby successful bisulfite conversion 17 d) Fast results • Competitor’s bisulfite kits contain aliquoted bisulfite salt that first needs to be dissolved and has a limited shelf life • Furthermore, some kits require an overnight conversion process • The EpiTect Fast Kits contain a ready-to-use bisulfite solution that can be stored at room temperature (15–25°C) for at least 6 months. • Bisulfite conversion in as short as 30 minutes of processing time with a conversion rate <99.8%
- 18. Sample to Insight 3)Challenges of bisulfite conversion Accurate DNA methylation analysisby successful bisulfite conversion 18 e) High recovery and concentration of bsDNA for downstream analysis Bisulfite treated DNA can be used for various downstream applications, e.g.: • PCR: end-point, real-time, and methylation-specific • Bisulfite sequencing (direct and cloning) • COBRA (Combined Bisulfite Restriction Analysis) • Pyrosequencing • Next Generation Sequencing Reliable downstream analysis required sufficient amount of intact bsDNA. EpiTect Fast Kits enable accurate downstream analysis by: • ensuring optimal DNA protection during bisulfite conversion • providing highly concentrated bsDNA in as little as 10 µl Elution Buffer
- 19. Sample to Insight 3)Challenges of bisulfite conversion Accurate DNA methylation analysisby successful bisulfite conversion 19 e) High recovery and concentration of bsDNA for downstream analysis • Detection technologies get more and more sensitive, but not necessarily more robust • Time and costs wasted for the processing of low-quality samples • The quantity of bisulfite-converted DNA can be determined using a spectrophotometer • Use a value of 40 µg/mL for A260 = 1.0, because the converted DNA more resembles RNA • Check DNA quality by determining the A260/A280 ratio of the bisulfite converted DNA, the ratio should be between 1.7 and 1.9 DNA purification Bisulfite treatment Amplification Analysis QC
- 20. Sample to Insight Agenda AccurateDNA methylation analysisby successful bisulfite conversion 20 Introduction to DNA methylation1 19 Analysis of DNA methylation2 20 Challenges of bisulfite conversion3 21 Overview of QIAGEN solutions for bisulfite conversion 4 22 Summary5 23
- 21. Sample to Insight 4)Overview of QIAGEN solutions for reliable bisulfite conversion Accurate DNA methylation analysisby successful bisulfite conversion 21 Product Key Advantages New features EpiTect Fast DNA Bisulfite Kit Highly concentrated DNAsuitable for all downstreamapplications; 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 LyseAllBisulfite Kit Minimized loss of sample material due to integrated sample lysis; high DNA concentration enables use in all downstreamapplications. 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 DNAfrom as little as one FFPE slice. Optimized DNA isolation, ready-to-use bisulfite solution and fast conversion time and low elution volume combined with proven DNA protection.
- 22. Sample to Insight Agenda AccurateDNA methylation analysisby successful bisulfite conversion 22 Introduction to DNA methylation1 19 Analysis of DNA methylation2 20 Challenges of bisulfite conversion3 21 Overview of QIAGEN solutions for bisulfite conversion 4 22 Summary5 23
- 23. Sample to Insight 5)Summary Accurate DNA methylation analysisby successful bisulfite conversion 23 EpiTect Fast Bisulfite Kits Features • Bisulfite conversion in as little as 30 minutes • Ready-to-use bisulfite conversion reagent • Unique DNA Protect Buffer to prevent DNA degradation during treatment • Integrated DNA isolation and conversion • Minimal elution volume to achieve highly concentrated bsDNA Benefits • Methylation analysis ready in just one day, no over-night incubations – saves time and allows faster results • No aliquoted bisulfite salt with limited shelf life, no dissolving steps – saves preparation time, material and money • Minimal DNA degradation – ensures higher yields of DNA can be analyzed and increases the likelihood of finding an intact DNA fragment for PCR analysis
- 24. Sample to Insight EpiTectFast System Accurate DNA methylation analysisby successful bisulfite conversion 24 Odering information • EpiTect Fast Bisulfite Kit (10) 59802 o Universal trial kit - DNA isolation and bisulfite conversion for all kinds of samples • EpiTect Fast DNA Bisulfite Kit o EpiTect Fast DNA Bisulfite Kit (50) 59824 o EpiTect Fast DNA Bisulfite Kit (200) 59826 o EpiTect Fast 96 Bisulfite Kit (2) 59720 • EpiTect Fast FFPE Bisulfite Kit o EpiTect Fast FFPE Bisulfite Kit (50) 59844 o EpiTect Fast FFPE Bisulfite Kit (200) 59846 o EpiTect Fast 96 FFPE Bisulfite Kit (2) 59740 • EpiTect Fast LyseAll Bisulfite Kit o EpiTect Fast LyseAll Bisulfite Kit (50) 59864 o EpiTect Fast LyseAll Bisulfite Kit (200) 59866 o EpiTect Fast 96 LyseAll Bisulfite Kit (2) 59760 EpiTect Fast BisulfiteSystem
- 25. Sample to Insight Questions? AccurateDNA methylation analysisby successful bisulfite conversion 25 Thank you very much for your attention! Dr. Inga Irle Inga.Irle@qiagen.com Contact QIAGEN Technical Service Call: 1-800-426-8157 for US Call: +49 2103-29-12400 for EU Email: techservice-na@qiagen.com techservice-eu@qiagen.com Qiawebinars@qiagen.com