EpiTect Bisulfite Kits

For complete bisulfite conversion and cleanup of DNA for methylation analysis

Features

  • 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 Kit (48)

Cat. No. / ID: 59104

48 EpiTect Bisulfite Spin Columns, Reaction Mix, DNA Protect Buffer, Carrier RNA, Buffers
₩347,000.00
Column typePlate type
Spin Column
96 well
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EpiTect Bisulfite Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

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 Connect.

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.

See figures

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").

See figures

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").

 

See figures

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

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsMultiplex PCR, real-time PCR, sequencing
ProcessingManual (spin and vacuum)
Sample typesBlood, (FFPE) tissue, DNA samples
Conversion efficiency99.4–99.8%
FormatSpin column and 96-well plate
Time per run or per prep<6 hours (1-48 samples), ~ 7 hours (48-96 samples)
Elution volumeVaries
Sample amount1–2 µg
YieldDepends on input amount of purified genomic DNA used for conversion

Resources

Analysis Software (5)
BRAF Plug-in 1.3
SOFTWARE (1MB)
Version 1.3.0.7
For use with PyroMark Q24 Software version 2.0.8
EGFR Plug-in 1.3
SOFTWARE (2MB)
Version 1.3.0.7
For use with PyroMark Q24 Software version 2.0.8
GIST Plug-in 1.3
SOFTWARE (1MB)
Version 1.3.0.7
For use with PyroMark Q24 Software version 2.0.8
KRAS Plug-in 1.3
SOFTWARE (1MB)
Version 1.3.0.6
For use with PyroMark Q24 Software version 2.0.8
RAS Extension Plug-in 1.3
SOFTWARE (1MB)
Version 1.3.0.7
For use with PyroMark Q24 Software version 2.0.8
Kit Handbooks (2)
For complete bisulfite conversion and cleanup of DNA for methylation analysis in 96-well format
Brochures & Guides (1)

FAQ

What are the expected PCR results when using EpiTect Control DNA on untreated or bisulfite-converted DNA?

Expected PCR results when using the EpiTect Control DNA Set in combination with the EpiTect MSP Kit can be found in the table below:

 

Type of DNA Primer for unmethylated & unconverted genomic DNA Primer for unmethylated & bisulfite converted target DNA Primer for methylated & bisulfite converted target DNA
Unmethylated & untreated control DNA PCR product NO PCR product NO PCR product
Unmethylated & bisulfite converted control DNA NO PCR product PCR product NO PCR product
Methylated & bisulfite converted control DNA NO PCR product NO PCR product PCR product
No template control NO PCR product NO PCR product NO PCR product

 

 

 

FAQ ID -2011
What happens when using EpiTect MethyLight Assays on only partially bisulfite converted DNA template?

If only two of three CpG sites are methylated in the template DNA, EpiTect MethyLight Assays used with the EpiTect MethyLight PCR Kit will detect the site as methylated, irrespective of conversion success for the third CpG site.

However, if only one of three CpG sites is methylated, and the remaining two unmethylated C residues fail to convert to U after bisulfite treatment, the EpiTect MethyLight System will detect the unmethylated site as methylated. Therefore we strongly recommend to use our EpiTect Bisulfite Kits to achieve 100% conversion rate.

 

 

FAQ ID -2009
After bisulfite conversion, can the DNA be stored?
Yes, DNA converted with EpiTect Bisulfite Kits and EpiTect Plus Bisulfite Kits is very stable and can be stored at -20 C for at least 3 years without decrease in quality or conversion. See the website for more details and data:  http://www.qiagen.com/products/epigenetics/epitect/epitectbisulfitekit.aspx#Tabs=t1
FAQ ID -2409
What are typical yields of bisulfite converted DNA when using the EpiTect Plus Bisulfite Kits?

The typical yields of bisulfite converted DNA when using the EpiTect Plus Bisulfite Kits depend on the amount of DNA and source of the starting material. Typically over 80% of DNA can be recovered after bisulfite conversion using EpiTect Plus DNA Kit.

Input amounts are as follows:

DNA: 1ng-2µg (EpiTect Plus DNA Bisulfite Kit)

Cells: 10-10E5 (EpiTect Plus LyseAll Bisulfite Kit)

Blood:0.5-20 µl (EpiTect Plus LyseAll Bisulfite Kit)

FFPE tissue:1 slice (10µm thick) (EpiTect Plus FFPE Bisulfite Kit)

FAQ ID -2408
How does the DNA Protection work in the EpitTect Bisulfite kits?

The DNA Protect buffer included in all EpiTect Bisulfite and EpiTect Plus Bisulfite kits works in two ways.

1) it serves as a scavenger for free radicals which are built during bisulfite conversion. As these free radicals can harm and fragment DNA the scavenger protects DNA from excessive degradation.

2) it keeps DNA single stranded even at lower temperatures so that DNA doesn’t need to be subjected to high temperatures for long time which is known to degrade DNA. Thus, it ensures high conversion rates even at gentle conversion conditions.

This is of special interest if working with low DNA qualities e.g. when working with formalin-fixed paraffin-embedded tissue samples.

FAQ ID -2410
What are the ideal and the largest PCR amplicon sizes when using the EpiTect MSP Kit?

An ideal amplicon size for bisulfite converted DNA (e.g., using EpiTect Bisulfite Kits) is up to 250 bp. However, we amplified fragments up to 700 bp successfully using bisulfite converted DNA as a template for the EpiTect MSP Kit. Note that the time for the elongation step needs to be increased for such long amplicons.

 

FAQ ID -2004
How long can DNA converted with EpiTect Bisulfite Kits be stored before use in methylation specific PCR with the EpiTect MSP Kit?

We have data for DNA converted with EpiTect Bisulfite Kits showing that the bisulfite treated DNA is stable for 3 years at -20°C before use in methylation specific PCR with the EpiTect MSP Kit, or the EpiTect MethyLight PCR Kit.

 

 

FAQ ID -2002
3345 - What is the difference between the high and low concentration EpiTect Bisulfite Conversion protocol?

The lower limit of both protocols is identical but always uses the high concentration protocol if possible. The low concentration protocol allows for more DNA to be used by eliminating some of the protection buffer.

If the concentration of this DNA is sufficiently high (1ng or 100ng DNA) in 20µl, always use the high concentration protocol.  If the DNA has a lower concentration, 40µl of DNA solution can be used at the expense of reducing the volume of Protect buffer (from 35 to 15µl). Because of this reduced protect buffer, the upper limit of input DNA for low concentrated samples is reduced from 2µg to 500ng to ensure consistently high DNA protection.

This is also true for the new EpiTect Fast, where we have identical instructions in the handbook.
FAQ - 3345
3343 - Why is it difficult to quantify DNA using UV/Vis spectroscopy and fluorescence methods following EpiTect Bisulfite Conversion? How best do you quantify it?

Bisulfite converted DNA is difficult to quantify by spectroscopic methods.  No longer double stranded, PicoGreen and ethidium bromide cannot be used as they only bind reliably to ds DNA.  Single standed DNA uses a extinction coefficient factor of 40 to calculate DNA concentrations from OD 260 readings however the factors for each base is also different as U has a very different extinction coefficient factor than C.  This would therefore require a known sequence of C and an estimate of CpG.  However, the larger concern is the residual radical scavenger used to protect the DNA distorts the absorbance at 260 nm.  However, this chemical does not cause any inhibition of PCR.

QIAGEN recommends quantifying bisulfite treated DNA by real time PCR using methods developed for bisulfite treated DNA.  Highly concentrated bisulfite DNA (>100 ng/ul) can be roughly determine by spectophotometric measurement.
FAQ - 3343
3336 - Can I use the linearized DNA for Epitect Bisulfite treatment with the Epitect Bisulfite kit?

Linearization per se is not a problem. With regards to circular DNA with a size below 20kb, linearization is even beneficial.

What is the purpose of the unmethylated and unconverted control DNA of the EpiTect PCR Control DNA Set?

The unmethylated and unconverted human control DNA of the EpiTect PCR Control DNA Set allows to check that primers designed for the specific detection of unmethylated and converted DNA (U-converted DNA), and for methylated, converted DNA (M-converted DNA) do not bind to untreated genomic DNA.*

In case bisulfite conversion was not complete, leaving certain unmethylated C residues unconverted, false positives would result if the primer specific for M-converted DNA binds to untreated gDNA.

This control DNA can also be used to check conversion efficiency during bisulfite treatment.

 

*(Summary of principle: Methylation of DNA occurs on cytosine residues, especially on CpG dinucleotides enriched in small regions of DNA. Incubation of target DNA with sodium bisulfite, using e.g. EpiTect Bisulfite Kits, results in conversion of unmethylated cytosine residues into uracil, leaving methylated cytosines unchanged).

 

FAQ ID -2007
3335 - Can I use the DNA directly from enzymatic reaction for Epitect Bisulfite treatment with the Epitect Bisulfite kit (cat. no. 59104)?

The depurination of DNA as the first step of the Epitect Bisulfite kit (cat. no. 59104) workflow is a chemical reaction, there's no problem at all using the DNA directly after enzymatic reaction.