EpiTect MethyLight PCR Kits

探针法定量real-time PCR分析甲基化状态

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EpiTect MethyLight PCR +ROX Vial Kit (200)

Cat. No. / ID:  59496

Master Mix without ROX for methylation-specific real-time PCR analysis, 200 x 50 µl reactions
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EpiTect MethyLight PCR 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

  • 基于TaqMan探针的real-time分析,准确度高
  • 配合MethyLight Assays,可靠进行甲基化定量检测
  • 方便的预混液形式,无需进一步优化

Product Details

EpiTect MethyLight PCR Kit和EpiTect MethyLight PCR + ROX Vial Kit确保高灵敏度的可靠定量检测,使用TaqMan探针或双标探针以区分甲基化位点。该试剂盒为即用型试剂盒,无需进一步优化。该试剂盒与各种定量PCR仪兼容,EpiTect MethyLight PCR Kit适用于除Applied Biosystems 7500以外的所有Applied Biosystems的PCR仪。EpiTect MethyLight PCR +ROX Vial Kit适用于所有其他的PCR仪,包括Applied Biosystems 7500和Bio-Rad、Cepheid、Corbett、Eppendorf、Roche和Stratagene的仪器。

Performance

EpiTect MethyLight PCR Kit可灵敏检测DNA甲基化状态的微小变化(参见" Sensitive real-time PCR quantification"),需与双标分析配合使用,如预设计的EpiTect MethyLight Assays。
See figures

Principle

EpiTect MethyLight PCR Kits与EpiTect MethyLight Assays或其他双标探针-引物配合使用,可灵敏定量亚硫酸氢盐转化后DNA中的甲基化DNA。根据对于序列甲基化状态区分程度的要求,MethyLight可用于定量和半定量分析。

双标记探针,包括TaqMan探针,是序列特异性的寡聚核苷酸,带有荧光基团和淬灭基团。荧光基团在探针的5'末端,而淬灭基团通常在3'末端或中间。在PCR的延伸阶段,Taq DNA聚合酶的5'→3'外切酶活性水解探针,将荧光基团和淬灭基团分离。可检测的荧光量与扩增的PCR产物成正比。

特异性甲基化探针定量MethyLight real-time PCR产物

使用MethyLight PCR进行定量甲基化分析,含有不同荧光标签的两个双标探针与一组PCR引物配合使用。亚硫酸氢盐转化后DNA需要一个不同的探针,以区分甲基化和非甲基化CpG位点。这些引物特异性针对非甲基化位点。一个探针在某一位点与甲基化DNA特异性结合,而其他探针特异性结合同一位点的非甲基化DNA。不同的探针包含不同的荧光基团,由此定量甲基化和非甲基化序列(参见" Quantitative MethyLight real-time PCR")。

特异性甲基化探针半定量MethyLight real-time PCR产物

对于半定量MethyLight分析,一个双标探针与一组特异性结合某些甲基化位点的PCR引物配合使用。亚硫酸氢盐转化后DNA需要一个不同的探针,以区分甲基化和非甲基化CpG位点,这些引物要特异性结合甲基化的亚硫酸氢盐转化后DNA。如果甲基化特异性引物结合到DNA上,它会在延伸阶段加长,并且Taq DNA Ploymerase的5'→3'外切酶活性会引起引物降解,释放荧光基团。由于荧光基团与淬灭基团已经隔离,可检测到荧光(参见" Semiquantitative MethyLight real-time PCR")。

提高特异性

EpiTect MethyLight Master Mix专用于序列特异性探针法高灵敏地检测甲基化和非甲基化DNA。除各种盐类和添加剂,缓冲液含有经优化的KCl和(NH4)2SO4组合,提高每个PCR扩增退火过程特异性引物与探针结合的比例。可确保区别探针杂交和检测非甲基化和/或甲基化靶序列。严谨的引物退火条件可在亚硫酸氢盐转化后DNA扩增过程中提高PCR特异性,能够可靠的进行基于探针的甲基化状态检测。

MethyLight PCR的对照反应

为确保用于检测甲基化或非甲基化DNA的MethyLight PCR探针和引物具有特异性,需要进行对照反应。应用不同浓度的亚硫酸氢盐转化后对照DNA(完全甲基化和完全非甲基化)。此外,确定甲基化程度过程中可将这些对照DNA混合物作为定量分析标准。配合EpiTect Control DNA Set,QIAGEN以即用型试剂盒的形式提供所有的对照DNA,包括:甲基化的亚硫酸氢盐转化后DNA、非甲基化的亚硫酸氢盐转化后DNA和人类基因组DNA,用于可靠的、标准化的甲基化特异性对照反应。

See figures

Procedure

EpiTect MethyLight PCR Kit以预混液的形式提供,确保快速方便的反应体系构建。提供两种试剂盒,即预混液中添加ROX和未添加ROX,针对各种不同的real-time循环仪可灵活选择。经验证的操作流程适用于大部分real-time循环仪,确保最佳效果。

EpiTect MethyLight PCR Kit提供含有ROX参比染料的预混液,专为配合需要高浓度ROX染料进行荧光校准的real-time PCR仪进行了优化(如Applied Biosystems的仪器,除了Applied Biosystems 7500 Real-Time PCR Systems)。

EpiTect MethyLight PCR + ROX Vial Kit提供不含ROX染料的预混液,但提供单独包装的ROX染料,可根据real-time循环仪的要求加入反应体系。该试剂盒用于需要较低浓度ROX染料进行荧光校准的循环仪(如Applied Biosystems 7500 Real-Time PCR Systems),可选择性地使用需ROX染料的循环仪(如Stratagene的仪器),以及无需ROX染料的仪器。

表观遗传学中标准化的工作流程

生物学和医药研究的很多领域中,尤其是肿瘤学、干细胞研究和发育生物学研究中,获得表观遗传学信息是首要任务。但是,由于缺乏提供可重复性数据的标准化方法,尤其是样本材料的限制,分析DNA的甲基化变化非常困难。QIAGEN最新推出的EpiTect方案,建立了从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甲基化分析或测序的标准化预分析和分析方案(参见" Standardized workflows in epigenetics")。

See figures

Applications

EpiTect MethyLight PCR Kits适用于定量和半定量real-time PCR分析,可与EpiTect MethyLight Assays或其他双标探针-引物配合使用。

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsQuantification of bisulfite converted DNA
Reaction typeHighly accurate and sensitive probe-based MethyLight real-time analysis
Real-time or endpointReal-time
With or without ROXAvailable with ROX in master mix and with ROX as separate vial
Sample/target typeBisulfite converted DNA
SYBR Green I or sequence-specific probessequence-specific probes
Single or multiplexSingle & duplex
Thermal cyclerMost real-time cyclers excepted Roche LightCycler®

Resources

试剂盒操作手册 (1)
EpiTect MethyLight PCR Kit
EpiTect MethyLight PCR + ROX Vial Kit
For quantitative methylation analysis using
sequence-specific probe-based real-time PCR
产品介绍与指南 (1)
Now with even more applications!
安全数据表 (1)
Download Safety Data Sheets for QIAGEN product components.
Certificates of Analysis (1)
Brochures & Guides (1)
Now with even more applications!
Kit Handbooks (1)
EpiTect MethyLight PCR Kit
EpiTect MethyLight PCR + ROX Vial Kit
For quantitative methylation analysis using
sequence-specific probe-based real-time PCR

FAQ

What is the advantage of EpiTect MethyLight PCR Kits over QuantiTect Probe PCR Kits for methylation studies?

The EpiTect MethyLight PCR Kit chemistry is especially developed for the requirements of bisulfite converted DNA. As a result of methylation and the conversion of unmethylated cytosines through bisulfite treatment, probe hybridization behavior differs from that of untreated DNA, and requires special buffer conditions. The balanced cations as well as Factor MP in the MethyLight PCR chemistry are contributing to the special hybridization behavior required for bisulfite converted DNA.

 

 

FAQ ID -2010
Can the EpiTect PCR Control DNAs also be used as standard in real-time methylation specific quantification?

Yes, the EpiTect Control DNA and Control DNA Set can be used as standards in real-time methylation specific quantification. For data, please see our EpiTect MethyLight PCR Product Profile.

 

 

 

FAQ ID -2006
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
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