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[Bio101] qPCR of Yeast ChIP DNA
[Bio101] 酵母ChIP-qPCR技术   

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Abstract

This protocol is adapted from Chris Yellman's personal notes for qPCR of yeast ChIP DNA to verify binding sites already identified by microarray/sequencing analysis. In addition, qPCR is a quick way to assay the quality of a ChIP sample (provided you have a few well-characterized binding sites) before submitting it for sequencing/microarray analysis. This protocol is designed for use with a Roche LightCycler.

Materials and Reagents

  1. PCR primers
    a.  Prepare 10 µM primer stocks in water from 100 µM stocks in TE.
    b.  Pick several positive control primer pairs and at least one negative site. Primer design criteria are described in the protocol.
  2. LightCycler 480 SYBR Green I Master mix (Roche Diagnostics, catalog number: 04707 516001 )
    The mix contains Taq, dNTPs and the intercalating SYBR Green dye.
  3. Yeast genomic DNA for a positive control dilution series
    You can use a glass bead lysis yeast genomic DNA preparation that has been RNase treated, Qiagen cleaned and stored in TE, or purchase directly from Invitrogen.
  4. 10 µl capacity multi-well pipetter
    384-well reaction plate with transparent adhesive plastic cover

Equipment

  1. Covered container
  2. Microcentrifuge tube
  3. 384-well reaction plate

Procedure

  1. Design PCR primers
    Primer 3 program (http://frodo.wi.mit.edu/primer3/) can be used to design primers. The following primer design criteria have worked for me: amplified regions of 200-250 bp in length, melting temperatures of ~59-61 °C and primer length 20. Other design criteria were chosen by Primer 3 at the default values.
  2. Prepare a positive control DNA dilution series
    To determine the amplification efficiency of each primer pair to be used, prepare a DNA dilution series. An 8-fold dilution series provides a convenient reference. Dilute the DNA in pure water from 8x diluted out to 87 (2,097,152x) diluted. Include a no DNA sample.
  3. Dilute the ChIP DNA samples in water
    The ChIP DNA must be diluted to an appropriate concentration. Each qPCR reaction will use 2 µl of dilute template.
    This amount is 1/40 of a ChIP from ~4.5 x 109 cells and it will suffice for 10 qPCR reactions with 1 µl left over. The ChIP DNA analyzed in each qPCR react ion represents the amount recovered from ~1 x 107 cells.
    Note: It's possible that this is too little ChIP DNA and the amount should be increased two-fold.
  4. Prepare the reaction mixtures (reaction volume of 10 µl)
    Prepare for triplicate (if the data are to be published) or duplicate analysis of each sample. I usually prepare enough volume for one extra reaction per 8-10 reactions, storing everything on ice and in a covered container (dark) during preparation.
    Each 10 µl reaction contains the following:
    a.  2 µl pure water
    b.  5 µl SYBR Green I Master
    c.  1 µl of PCR primers (0.5 µl of each primer in a pair from 10 µM stock)
    d.  2 µl DNA template
    First dilute the SYBR Green I Master mix with 2 µl of pure water + 5 µl of SYBR Green per reaction. Distribute the desired volume of dilute SYBR Green master mix into a microcentrifuge tube for each primer pair. Add the PCR primers (remember to include the extra reaction volume) to prepare primer pair master mixes.
  5. Set up the qPCR reactions and add templates
    Distribute 8 µl of the desired master reaction mixture to each well of a 384-well reaction plate. Add 2 µl of template DNA to each well and water to the negative controls and cover the plate with clear adhesive plastic film. Keep in the dark until running the PCR.
  6. Run the qPCR thermocycler program
    In addition to the qPCR sample reactions, set up melting curve analysis for each primer pair used.
  7. qPCR data analysis
    Verify that the melting curve analysis returns a single peak for each primer pair. Determine relative quantification by the 2-ΔΔCp method (2nd derivative maximum) and calculate the mean ΔΔCp value for each set of duplicate or triplicate samples. For each target tested, calculate the standard deviation of the mean ΔΔCp across a panel of negative controls. Determine the statistical significance of the data assuming a normal distribution. Note: One modification to relative quantification by the 2-ΔΔCp method would be to apply an efficiency-corrected equation (Pfaffl, 2001).

Notes

Quantitative PCR (qPCR) uses fluorescence to detect PCR product accumulation. The crossing point (Cp) or threshold cycle (Ct) is the point at which fluorescence rises appreciably above background. Other methods of analysis use the linear range of the amplification curve. The Cp value is used for the relative quantitation of qPCR. The qPCR machine detects the fluorescence and software calculates Cp values from the intensity of the fluorescence. I have used the method of maximum second derivative of the amplification curve as a standard to compare samples. This is the point on the curve with the maximum positive change in curvature. It is the point at which the signal becomes detectable and enters the linear range of amplification.
The SYBR Green method uses a dye in the PCR reaction which binds to newly synthesized double-stranded DNA and emits fluorescence The SYBR Green dye intercalates with doublestranded DNA, cansing the SYBR Green to fluoresce.
Real-time PCR, also known as kinetic PCR, qPCR, qRT-PCR and RT-qPCR, is a quantitative PCR method for the determination of copy number of PCR templates such as DNA or cDNA in a PCR reaction. There are two types of real-time PCR: probe-based and intercalator-based. Both methods require a special thennocycler equipped with a sensitive camera that monitors the fluorescence in each well of the multi-well plate at frequent intervals during the PCR Reaction. Probe-based real-time PCR, also known as TaqMan PCR, requires a pair of PCR primers asregular PCR does, an additional fluorogenic probe which is an oligonucleotide with both a reporter fluorescent dye and a quencher dye attached. The TaqMan method is more accurate and reliable than the SYBR Green method, but also more expensive.

References

  1. Bustin, S. A., Benes, V., Nolan, T. and Pfaffl, M. W. (2005). Quantitative real-time RT-PCR--a perspective. J Mol Endocrinol 34(3): 597-601.
  2. Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9): e45.
  3. Tichopad, A., Dilger, M., Schwarz, G. and Pfaffl, M. W. (2003). Standardized determination of real-time PCR efficiency from a single reaction set-up. Nucleic Acids Res 31(20): e122.

简介

该协议改编自Chris Yellman的关于酵母ChIP DNA的qPCR的个人笔记,以验证已通过微阵列/测序分析鉴定的结合位点。 此外,qPCR是在提交用于测序/微阵列分析之前测定ChIP样品的质量的快速方法(前提条件是具有一些充分表征的结合位点)。 此协议设计用于Roche LightCycler。

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材料和试剂

  1. PCR引物
    a。  在TE中制备100μM储备液中的10μM引物储备液 b。  挑选几个阳性对照引物对和至少一个阴性位点。 底漆设计标准在协议中描述。
  2. LightCycler 480 SYBR Green I Master mix(Roche Diagnostics,目录号:04707 516001)
    该混合物含有Taq,dNTP和插入的SYBR Green染料
  3. 阳性对照稀释系列的酵母基因组DNA 您可以使用经RNase处理的玻璃珠裂解酵母基因组DNA制备物,Qiagen清洗并保存在TE中,或直接从Invitrogen购买。
  4. 10μl容量多孔移液器
    384孔反应板,带透明粘性塑料盖

设备

  1. 覆盖容器
  2. 微量离心管
  3. 384孔反应板

程序

  1. 设计PCR引物
    Primer 3计划( http://frodo.wi.mit.edu/primer3/)可以 用于设计引物。 以下引物设计标准适用于我:200-250bp的扩增区域 长度,熔融温度〜59-61℃和引物长度20.其他设计标准由底漆3选择默认值。
  2. 准备阳性对照DNA稀释系列
    为了确定使用的每个引物对的扩增效率,制备DNA稀释系列。 8倍稀释系列提供了方便的参考。稀释DNA在纯水从8x稀释至87(2,097,152x)稀释。包括无DNA样品。
  3. 将ChIP DNA样品在水中稀释
    ChIP DNA必须稀释至适当的浓度。每个qPCR反应将使用2μl稀释模板 这个量是从〜4.5×10 9个细胞的ChIP的1/40,并且其足以用于剩余1μl的10个qPCR反应。在每个qPCR反应离子中分析的ChIP DNA代表从〜1×10 7个细胞回收的量。
    注意:这可能是ChIP DNA太少,数量应该增加两倍。
  4. 准备反应混合物(反应体积10μl)
    准备一式三份(如果要发布数据)或每个样品的重复分析。我通常准备足够的体积,每8-10反应一个额外的反应,存储一切在冰上和在一个有盖的容器(黑暗)在准备期间。
    每10μl反应含有以下内容:
    a。  2μl纯水
    b。  5μlSYBR Green I Master
    c。  1μlPCR引物(0.5μl来自10μM储备物的一对引物) d。 2μlDNA模板
    首先用2μl纯水+每次反应5μlSYBR Green稀释SYBR Green I Master混合物。将所需体积的稀释SYBR Green主混合物分配到每个引物对的微量离心管中。加入PCR引物(记住包括额外的反应体积)以制备引物对主混合物
  5. 设置qPCR反应和添加模板
    将8μl所需的主反应混合物分配到384孔反应板的每个孔中。加入2微升的模板DNA到每个孔和水到阴性对照,并用透明粘性塑料膜覆盖板。保持在黑暗中,直到运行PCR。
  6. 运行qPCR热循环仪程序
    除了qPCR样品反应之外,对使用的每个引物对设置解链曲线分析。
  7. qPCR数据分析
    验证熔解曲线分析为每个引物对返回单个峰。通过2- ΔΔ Cp方法(二阶导数最大值)确定相对定量,并计算每组一式两份或三份样品的平均ΔΔ Cp值。对于每个测试的目标,计算阴性对照组中的平均值的标准偏差ΔΔp Cp。确定数据的统计显着性,假设正态 分配。注意:通过2- ΔΔ Cp方法的相对定量的一个修改将是应用效率校正方程(Pfaffl,2001)。

笔记

定量PCR(qPCR)使用荧光检测PCR产物积累。交叉点(Cp)或阈值循环(Ct)是荧光上升明显高于背景的点。其他分析方法使用扩增曲线的线性范围。 Cp值用于qPCR的相对定量。 qPCR机器检测荧光,并且软件根据荧光的强度计算Cp值。我已经使用扩增曲线的最大二阶导数的方法作为比较样品的标准。这是曲线上具有曲率最大正变化的点。它是信号变得可检测并进入放大线性范围的点 SYBR Green方法在PCR反应中使用染料,其结合新合成的双链DNA并发射荧光SYBR Green染料插入双链DNA,使SYBR Green发出荧光。
实时PCR,也称为动力学PCR,qPCR,qRT-PCR和RT-qPCR,是用于测定PCR反应中PCR模板(例如DNA或cDNA)的拷贝数的定量PCR方法。有两种类型的实时PCR:基于探针的和基于嵌入剂的。两种方法都需要配备有灵敏摄像机的特殊的循环器,其在PCR反应期间以频繁的间隔监测多孔板的每个孔中的荧光。基于探针的实时PCR,也称为TaqMan PCR,需要一对PCR引物作为常规PCR,另外的荧光探针是具有报道荧光染料和猝灭染料的寡核苷酸。 TaqMan方法比SYBR Green方法更准确和可靠,但也更昂贵。

参考文献

  1. Bustin,S.A.,Benes,V.,Nolan,T。和Pfaffl,M.W。(2005)。 定量实时RT-PCR - 透视。 Endocrinol 34(3):597-601。
  2. Pfaffl,M.W。(2001)。 实时RT-PCR中相对定量的新数学模型 Nucleic Acids Res。 29(9):e45。
  3. Tichopad,A.,Dilger,M.,Schwarz,G。和Pfaffl,M.W。(2003)。 从单一反应设置标准化测定实时PCR效率。 em> Nucleic Acids Res 31(20):e122。
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zheng, W. (2011). qPCR of Yeast ChIP DNA. Bio-protocol Bio101: e135. DOI: 10.21769/BioProtoc.135;
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Bioseeker B
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I don't know how to design a primer. Could you please explain about it? Thanks.
7/29/2016 8:09:09 AM Reply