For complete bisulfite conversion and cleanup of DNA for methylation analysis
  • Streamlined procedure for fast and reliable results
  • Complete cytosine conversion
  • Unique DNA protection system for highly sensitive analysis
  • Optimized protocols, including conversion from FFPE DNA
  • Spin-column and 96-well plate formats for high throughput

EpiTect Bisulfite Kits enable complete conversion of unmethylated cytosines to uracils and subsequent purification in 6 hours. The EpiTect 96 Bisulfite procedure takes less than 7 hours. The highly sensitive method utilizes innovative protection against DNA degradation that guarantees sensitive results, even from 1 ng DNA, and ensures high conversion rates of over 99%. Due to its unique DNA Protect technology, EpiTect Bisulfite converted DNA is highly suitable for whole bisulfitome amplification using EpiTect Whole Bisulfitome Kits. This enables reliable amplification of DNA in cases where bisulfite converted DNA is limited. EpiTect Bisulfite Kits include a spin-column or 96-well format procedure, and can be processed using a centrifuge or vacuum manifold. In addition, the EpiTect Bisulfite Kit can be fully automated on the QIAcube.

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EpiTect Bisulfite Kit (48)
48 EpiTect Bisulfite Spin Columns, Reaction Mix, DNA Protect Buffer, Carrier RNA, Buffers
59104
$437.00
EpiTect 96 Bisulfite Kit (2)
2x EpiTect Bisulfite 96-well Plates, Reaction Mix, DNA Protect Buffer, Carrier RNA, Buffers
59110
$342.00

EpiTect Bisulfite Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.



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Long-term DNA storage.|Highly efficient cytosine conversion.|Complete cytosine conversion.|Unique DNA Protect Buffer and pH indicator system.|Methylation detection from FFPE tissue samples.|EpiTect Bisulfite Conversion procedure.|Fast and easy bisulfite conversion.|Time-saving procedure.|Standardized workflows in epigenetics.|
Bisulfite converted DNA generated with EpiTect Kits was stored at –20°C for up to 36 months, and amplified at several time intervals. The resulting threshold cycle values at all time points for various DNA amounts were comparable to those obtained with freshly converted DNA (time point 0).|Over 900 clones generated by PCR from 6 independent EpiTect Bisulfite reactions (1 μg DNA each) and cloned using the QIAGEN PCR Cloning Kit were sequenced. Over 150,000 of the 450,000 sequenced nucleotides were unmethylated cytosine residues (DNA 1) that were converted into uracil residues (DNA 2). Cytosine to uracil conversion rates of 99.4–99.8% were obtained. The sequence shown is a small portion of the total sequence analyzed. DNA 1: untreated DNA; DNA 2: converted DNA.|A 1 μg aliquot of genomic DNA was converted using the EpiTect Bisulfite Kit or bisulfite kits from alternative suppliers (Supplier E and Supplier Z) according to the manufacturer's instructions. Next, the presence of unconverted DNA in comparable amounts of each sample was determined by [A] end-point PCR using the HotStarTaq Plus Master Mix Kit and [B] SYBR® Green based real-time PCR using the QuantiTect SYBR® Green PCR Kit. The EpiTect Bisulfite Kit treated DNA was completely converted, while DNA treated using kits from Suppliers E and Z contained a significant proportion of unconverted DNA.|[A] DNA Protect Buffer prevents DNA fragmentation during bisulfite treatment of DNA and facilitates the formation of single-stranded DNA, enabling complete bisulfite conversion. [B] The pH indicator dye in the DNA Protect Buffer enables visualization of complete reaction mixing, thereby ensuring the correct pH is achieved for complete cytosine conversion.|DNA (1 µg) isolated from an FFPE sample using the QIAamp DNA Mini Kit was converted with the EpiTect Bisulfite Kit using the FFPE protocol. 1 μl of the 40 μl eluate was analyzed by [A] end-point PCR and [B] TaqMan probe-based real-time PCR. Both assays for the Glutathione S-Transferase gene, were specific for converted DNA. In each application the unconverted genomic DNA (control) was not amplified.||Human genomic DNA was purified from blood using the QIAamp DNA Blood Mini Kit, and various amounts (1 ng – 1 µg) were converted using the EpiTect Bisulfite Kit. PCR was performed using the HotStarTaq Master Mix Kit and 2 sets of primers designed to amplify converted DNA. A 5 µl aliquot of each PCR was loaded onto a 1.3% agarose gel. As little as 1 ng DNA is sufficient for conversion using the EpiTect Bisulfite Kit. C: untreated genomic DNA (negative control). M: marker.|The complete EpiTect Bisulfite Kit centrifugation or vacuum procedure — from DNA to pure bisulfite converted DNA ready for analysis — takes less than 6 hours. Traditional bisulfite conversion procedures can take over 18 hours and require extensive user interaction.|  |
Performance
Rapid results

The complete EpiTect Bisulfite Kit centrifugation or vacuum procedure — from DNA to pure bisulfite converted DNA ready for analysis — takes only 6 hours. The EpiTect 96 Bisulfite procedure takes under 7 hours. Traditional bisulfite conversion procedures can take over 18 hours and require extensive user interaction (see table "Fast and Easy Bisulfite Conversion" and figure "Time saving procedure"). The EpiTect Bisulfite Kit provides everything required for successful bisulfite conversion and DNA cleanup.

Fast and easy bisulfite conversion
  EpiTect procedure Traditional procedure
Reagent setup 10 minutes; prealiquoted solutions 40 minutes; reagent setup needed
Bisulfite reaction 5 hours 16 hours
Purification 30–45 minutes; EpiTect spin columns and plates 120 minutes; purification, desulfonation, precipitation, and washing steps


Complete DNA conversion

Each EpiTect Bisulfite Kit reaction converts 1 ng – 2 µg of DNA with equal efficiency. The novel procedure and optimized buffers enable the recovery of high yields of converted DNA. The highly efficient cytosine conversion is apparent by the low CT values obtained when amplifying converted DNA using real-time PCR (see figure "Complete cytosine conversion"). Analysis of EpiTect Bisulfite Kit converted DNA shows over 99% conversion of unmethylated cytosines (see figure "Highly efficient cytosine conversion"). This high conversion rate provides repeatable and dependable downstream analysis.

Principle
Bisulfite conversion of challenging samples

Due to the limited amount of DNA available, determination of methylation patterns in DNA from precious and limited sample materials (e.g., formalin-fixed paraffin-embedded [FFPE] or microdissected tissue) is especially challenging. However, such sample types are of specific interest for the successful identification of valuable disease-predicting biomarkers. EpiTect Bisulfite Kits address these challenges with optimized protocols for these sample types (see figure "Methylation detection from FFPE tissue samples"). All of the required buffers and reagents are provided, and only minimal handling is required.

For direct bisulfite conversion of DNA from FFPE samples, cells, blood, and tissue without prior DNA isolation, we recommend EpiTect Plus FFPE Bisulfite Kits and EpiTect Plus LyseAll Bisulfite Kits.

 Preventing DNA fragmentation

DNA Protect Buffer is uniquely formulated to prevent DNA fragmentation (usually associated with bisulfite treatment of DNA at high temperatures and low pH values) and provide effective DNA denaturation, resulting in the single-stranded DNA necessary for complete cytosine conversion (see "Unique DNA Protect Buffer and pH indicator system"). The prevention of fragmentation enables subsequent amplification and analysis of large PCR fragments (see figure "Fast and easy bisulfite conversion"). DNA Protect Buffer also contains a pH indicator dye, allowing confirmation of the correct pH for cytosine conversion. The efficient integrated DNA cleanup allows storage of the converted DNA for at least 12 months without affecting the DNA quality (see figure "Long-term DNA storage").

Procedure

The EpiTect procedure comprises a few simple steps, based on QIAGEN's straightforward bind, wash, and elute system (see flowchart "Conversion procedure"). After thermal denaturation and sodium bisulfite DNA conversion, the DNA is applied to an EpiTect spin column or plate where, using optimized buffers and a standard microcentrifuge or vacuum manifold, it is washed to remove all traces of sodium bisulfite and eluted. The eluted DNA can be used in all downstream applications, including methylation-specific PCR, real-time PCR analysis, bisulfite sequencing, COBRA, and Pyrosequencing.

Accessing epigenetic information is of prime importance for many areas of biological and medical research — particularly oncology, but also stem cell research and developmental biology. However, the analysis of changes in DNA methylation is challenging due to the lack of standardized methods for providing reproducible data, particularly from limited sample material. With its newly introduced EpiTect solutions, QIAGEN makes available standardized, pre-analytical and analytical solutions: from DNA sample collection, stabilization and purification, to bisulfite conversion and real-time or end-point PCR methylation analysis or sequencing (see figure "Standardized workflows in epigenetics").

 

Applications

DNA converted and purified using the EpiTect Bisulfite Kit is suitable for use in a range of downstream applications, including:

Methylation-specific PCR, real-time PCR
COBRA 
Sequencing/Pyrosequencing/NGS
High-resolution melting
Methylation arrays
Feature
Specifications
Applications Multiplex PCR, real-time PCR, sequencing
Conversion efficiency 99.4–99.8%
Elution volume Varies
Format Spin column and 96-well plate
Processing Manual (spin and vacuum)
Sample amount 1–2 µg
Sample types Blood, (FFPE) tissue, DNA samples
Time per run or per prep <6 hours (1-48 samples), ~ 7 hours (48-96 samples)
Yield Depends on input amount of purified genomic DNA used for conversion

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Kit Handbooks
4
メチル化解析のためのbisulfite法による完全なDNA変換およびクリーンアップ用
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DNA メチル化解析のためのBisulfite 処理による完全なDNA 変換とクリーンアップ (96ウェルフォーマット)
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For complete bisulfite conversion and cleanup of DNA for methylation analysis
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For complete bisulfite conversion and cleanup of DNA for methylation analysis in 96-well format
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