EpiTect Bisulfite Kits

亚硫酸氢盐的完全转化和DNA回收，用于甲基化分析

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EpiTect Bisulfite Kit (48)

Cat. No. / ID: 59104

48 EpiTect Bisulfite Spin Columns, Reaction Mix, DNA Protect Buffer, Carrier RNA, Buffers

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Column typePlate type

Spin Column

96 well

EpiTect Bisulfite Kits 旨在用于分子生物学应用。这些产品不能用于疾病诊断、预防和治疗。

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

简化的流程，获得快速、可靠的结果
胞嘧啶完全转化
独特的DNA保护体系，用于高度敏感的分析
优化的试验方案可处理FFPE组织样本DNA
离心柱和高通量96孔板方式

Product Details

EpiTect Bisulfite Kit可在6小时内将DNA 序列中非甲基化的胞嘧啶完全转化为尿嘧啶。EpiTect 96 Bisulfite操作时间少于7小时。高灵敏度的方法采用全新的保护机制防止DNA降解，确保即使是1 ng的DNA也可获得灵敏的结果，转化率可高达99%。得益于独特的DNA保护技术，EpiTect Bisulfite转化后DNA高度适用于EpiTect Whole Bisulfitome Kits转化后全基因组扩增。因此可在转化后DNA量较少的情况下确保可靠的扩增。EpiTect Bisulfite Kits包括离心柱或96孔板形式的扩增和脱磺酸基过程，确保快速可靠的实验结果用于下游应用。两个试剂盒都可用于离心机或真空装置，另外，EpiTect Bisulfite Kit可在QIAcube全自动核酸纯化仪上全自动运行。

Performance

快速得到实验结果

完整的EpiTect离心和真空过程只需6小时，包括从DNA到待分析的亚硫酸氢盐转化后的高纯DNA。EpiTect 96 Bisulfite操作时间少于7小时。传统的亚硫酸氢盐转化过程需要18个小时，且需要大量的操作(参见" 快速简单的亚硫酸氢盐转化"和" Time saving procedure")。EpiTect Bisulfite Kit为成功进行亚硫酸氢盐转化和DNA回收提供所需的全部试剂。

快速简单的亚硫酸氢盐转化
	EpiTect流程	传统流程
反应构建	10分钟；预制溶液	40分钟；需反应构建
亚硫酸氢盐转化	5小时	16小时
纯化	30–45分钟； EpiTect离心柱或孔板	120分钟；纯化、脱璜、沉淀和洗涤

完全的DNA转化

每个EpiTect Bisulfite Kit可以相同的效率转化低至1 ng到2 µg DNA。全新的实验步骤和优化的缓冲液可回收得到高产量的转化DNA。当通过real-time PCR扩增经转化的DNA时，低C_T值的实验结果显示了高效的胞嘧啶转化(参见" Complete cytosine conversion")。对转化的DNA的分析显示，EpiTect Bisulfite Kit可转化超过99%的非甲基化的胞嘧啶(参见" Highly efficient cytosine conversion")。高转化率确保了高重复性和可靠的下游分析结果。

See figures

Fast and easy bisulfite conversion.

Time-saving procedure.

Complete cytosine conversion.

Highly efficient cytosine conversion.

Principle

特殊样本的亚硫酸氢盐转化

从珍稀样本和少量样本（如：福尔马林固定、FFPE石蜡包埋或显微切割组织）中确定甲基化模式，由于DNA的量很少，实验尤其困难。但是这些样本类型是成功鉴定有价值的疾病，预测生物标记物的专门研究对象。 EpiTect Bisulfite Kit 为这些类型的样本提供优化的操作流程，从而解决了难题（参见" Methylation detection from FFPE tissue samples"）。该试剂盒含有实验所需的全部缓冲液和试剂，只需少量的手动操作。

对于从FFPE样本、细胞、血液及组织中直接进行亚硫酸氢盐转化而无需预先分离DNA，我们推荐使用EpiTect Plus FFPE Bisulfite Kit和EpiTect Plus LyseAll Bisulfite Kit。

防止DNA降解

DNA保护缓冲液采用独特的配方，防止DNA过度降解（在高温、低pH值条件下使用亚硫酸氢盐处理DNA时经常出现严重的DNA降解），并实现有效的DNA变性，从而产生完全转化为胞嘧啶时所需的单链DNA（参见" Unique DNA Protect Buffer and pH indicator system"）。防止DNA降解的发生可以保证后续大PCR片段的扩增和分析（参见" Fast and easy bisulfite conversion"）。DNA保护缓冲液含有PH指示剂染料，从而可保证用于胞嘧啶转化的正确的pH条件。有效的完整DNA回收可保证转化后的DNA至少保存12个月而不影响DNA的质量（参见" Long-term DNA storage"）。

See figures

Methylation detection from FFPE tissue samples.

Unique DNA Protect Buffer and pH indicator system.

Fast and easy bisulfite conversion.

Long-term DNA storage.

Procedure

EpiTect Bisulfite操作流程基于QIAGEN简单直接的结合、洗涤和洗脱体系，仅需几个简单的步骤（参见" Conversion procedure"）。DNA经过热变性和亚硫酸氢盐转化后，被上样到EpiTect离心柱或96孔板上，使用优化的缓冲液和标准化的离心步骤或真空装置，洗去所有的亚硫酸氢钠并洗脱DNA。洗脱的DNA可用于各种下游应用，如：甲基特异性PCR、real-time PCR分析、亚硫酸氢盐转化后测序（bisulfite sequencing）、结合亚硫酸氢盐的限制性内切酶法分析（COBRA）和焦磷酸测序（Pyrosequencing）。

表观遗传学信息对于生物医学研究（尤其是肿瘤学）、干细胞研究以及发育生物学等领域都非常重要。但是分析DNA甲基化的变化目前仍具有挑战性，因为缺乏标准化的方法可从有限的样本中得到可重现性的数据。QIAGEN推出新的表观遗传学解决方案，使样本分析前处理到分析整个过程实现标准化，即从DNA样本收集、稳定和纯化，到亚硫酸氢盐转化和real-time或终点式PCR甲基化分析或测序（参见" Standardized workflows in epigenetics"）。

See figures

EpiTect Bisulfite Conversion procedure.

Standardized workflows in epigenetics.

Applications

经EpiTect Bisulfite Kit转化和纯化的DNA适用于多种下游应用，如：

甲基化特异性PCR 、real-time PCR
结合亚硫酸氢盐的限制性内切酶法分析（COBRA）
测序/焦磷酸测序（Pyrosequencing）/NGS
高分辨率熔解
甲基化芯片

Supporting data and figures

Long-term DNA storage.

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).

Specifications

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

Resources

For complete bisulfite conversion and cleanup of DNA for methylation analysis in 96-well format

Download Safety Data Sheets for QIAGEN product components.

For complete bisulfite conversion and cleanup of DNA for methylation analysis in 96-well format

FAQ

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

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

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

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

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

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

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

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

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

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

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.