EpiTect Bisulfite Kits

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 Connect.

产品 货号 目录价:
EpiTect Bisulfite Kit (48)
48 EpiTect Bisulfite Spin Columns, Reaction Mix, DNA Protect Buffer, Carrier RNA, Buffers
59104
询价
EpiTect 96 Bisulfite Kit (2)
2x EpiTect Bisulfite 96-well Plates, Reaction Mix, DNA Protect Buffer, Carrier RNA, Buffers
59110
询价

EpiTect Bisulfite Kits适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。



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长期储存的DNA。|高效的胞嘧啶转化。|完全的胞嘧啶转化。|独特的DNA Protect Buffer和pH值指示体系。|对FFPE组织样本进行甲基化检测。|EpiTect Bisulfite Conversion实验方案。|快速、便利的亚硫酸氢盐转化。|省时的实验流程。|表观遗传学研究的标准工作流程。|
使用EpiTect Kits制备的亚硫酸氢盐转化DNA在–20°C下保存36个月,在几个时间段进行扩增。不同DNA量在不同时间点获得的阈值周期值都与新转化DNA(时间点为0)相当。|通过6个独立的EpiTect Bisulfite PCR反应(每个1 μg DNA)进行扩增,使用QIAGEN PCR Cloning Kit进行克隆,对获得的超过900个克隆进行测序。测序的450,000个核苷酸中有超过150,000个从未甲基化的胞嘧啶残基(DNA 1)被转化成尿嘧啶残基(DNA 2)。胞嘧啶转化成尿嘧啶的转化率达99.4–99.8%。展示的序列式总序列中的一小部分。DNA 1:未处理DNA;DNA 2:转化DNA。|根据操作指南,使用EpiTect Bisulfite Kit或其他供应商(Supplier E和Supplier Z)的亚硫酸氢盐转化试剂盒转化1 μg基因组DNA。然后,检测每个样本中未转化DNA的量,检测方法是[A]使用HotStarTaq Plus Master Mix Kit进行终点法PCR和[B]使用QuantiTect SYBR® Green PCR Kit进行SYBR® Green法real-time PCR。EpiTect Bisulfite Kit处理的DNA完全被转化,而使用Suppliers E和Z的试剂盒处理的DNA有很大比例的未转化DNA。|[A]在亚硫酸氢盐处理DNA时,DNA Protect Buffer可避免DNA片段化,便于单链DNA的形成,确保完全的亚硫酸氢盐转化。[B]DNA Protect Buffer中的pH指示剂使反应混合可视化,确保获得完全胞嘧啶转化所需的正确pH值。|使用QIAamp DNA Mini Kit从FFPE组织样本中分离DNA,再使用EpiTect Bisulfite Kit按照FFPE组织实验方案转化DNA(1 µg)。从40 μl洗脱液中取1 μl进行[A]终点法PCR和[B] TaqMan探针法real-time PCR。都检测在转化DNA中特异的Glutathione S-Transferase基因。每个应用中,未转化的基因组DNA(对照)未被扩增。||使用QIAamp DNA Blood Mini Kit从血液中纯化人类基因组DNA,使用EpiTect Bisulfite Kit转化不同量DNA(1 ng – 1 µg)。使用HotStarTaq Master Mix Kit进行PCR,设计2对引物扩增转化DNA。每个PCR反应取5 µl上样到1.3%琼脂糖凝胶上。使用EpiTect Bisulfite Kit可转化少至1 ng DNA。C: 未处理的基因组DNA(阴性对照)M: 分子量标准。|完整的EpiTect Bisulfite Kit离心或真空操作流程(从DNA到用于分析的纯化亚硫酸氢盐转化DNA)只需不到6小时。传统的亚硫酸氢盐转化流程需要超过18小时,且需要繁琐的实验操作。||
性能
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.

原理
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").

操作流程

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").

 

应用

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

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试剂盒操作手册
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For complete bisulfite conversion and cleanup of DNA for methylation analysis in 96-well format
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安全数据表
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Download Safety Data Sheets for QIAGEN product components.
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Superior storage stability of bisulfite converted DNA
长期储存的DNA。
使用EpiTect Kits制备的亚硫酸氢盐转化DNA在–20°C下保存36个月,在几个时间段进行扩增。不同DNA量在不同时间点获得的阈值周期值都与新转化DNA(时间点为0)相当。
Highly Efficient Cytosine Conversion
高效的胞嘧啶转化。
通过6个独立的EpiTect Bisulfite PCR反应(每个1 μg DNA)进行扩增,使用QIAGEN PCR Cloning Kit进行克隆,对获得的超过900个克隆进行测序。测序的450,000个核苷酸中有超过150,000个从未甲基化的胞嘧啶残基(DNA 1)被转化成尿嘧啶残基(DNA 2)。胞嘧啶转化成尿嘧啶的转化率达99.4–99.8%。展示的序列式总序列中的一小部分。DNA 1:未处理DNA;DNA 2:转化DNA。
Complete cytosine conversion
完全的胞嘧啶转化。
根据操作指南,使用EpiTect Bisulfite Kit或其他供应商(Supplier E和Supplier Z)的亚硫酸氢盐转化试剂盒转化1 μg基因组DNA。然后,检测每个样本中未转化DNA的量,检测方法是[A]使用HotStarTaq Plus Master Mix Kit进行终点法PCR和[B]使用QuantiTect SYBR® Green PCR Kit进行SYBR® Green法real-time PCR。EpiTect Bisulfite Kit处理的DNA完全被转化,而使用Suppliers E和Z的试剂盒处理的DNA有很大比例的未转化DNA。
Unique DNA Protect Buffer and pH indicator system
独特的DNA Protect Buffer和pH值指示体系。
[A]在亚硫酸氢盐处理DNA时,DNA Protect Buffer可避免DNA片段化,便于单链DNA的形成,确保完全的亚硫酸氢盐转化。[B]DNA Protect Buffer中的pH指示剂使反应混合可视化,确保获得完全胞嘧啶转化所需的正确pH值。
Methylation Detection from FFPE Tissue Samples
对FFPE组织样本进行甲基化检测。
使用QIAamp DNA Mini Kit从FFPE组织样本中分离DNA,再使用EpiTect Bisulfite Kit按照FFPE组织实验方案转化DNA(1 µg)。从40 μl洗脱液中取1 μl进行[A]终点法PCR和[B] TaqMan探针法real-time PCR。都检测在转化DNA中特异的Glutathione S-Transferase基因。每个应用中,未转化的基因组DNA(对照)未被扩增。
Epitect Bisulfite Conversion Procedure
EpiTect Bisulfite Conversion实验方案。
Fast and easy bisulfite conversion
快速、便利的亚硫酸氢盐转化。
使用QIAamp DNA Blood Mini Kit从血液中纯化人类基因组DNA,使用EpiTect Bisulfite Kit转化不同量DNA(1 ng – 1 µg)。使用HotStarTaq Master Mix Kit进行PCR,设计2对引物扩增转化DNA。每个PCR反应取5 µl上样到1.3%琼脂糖凝胶上。使用EpiTect Bisulfite Kit可转化少至1 ng DNA。C: 未处理的基因组DNA(阴性对照)M: 分子量标准。
Time saving procedure
省时的实验流程。
完整的EpiTect Bisulfite Kit离心或真空操作流程(从DNA到用于分析的纯化亚硫酸氢盐转化DNA)只需不到6小时。传统的亚硫酸氢盐转化流程需要超过18小时,且需要繁琐的实验操作。
Standardized Workflows in Epigenetics
表观遗传学研究的标准工作流程。