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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.

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

微生物学 > 微生物遗传学 > RNA > qRT-PCR
作者: Wei Zheng
Wei ZhengAffiliation: Keck Biotech Services, Yale University, New Haven, USA
For correspondence: wei.zheng.madison@gmail.com
Bio-protocol author page: a10
9/20/2011, 8482 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.135

[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.

[Abstract] 这个方法适用于Chris Yellman's personal notes ChIP DNA的定量PCR确定通过microarray或测序分析已经辨别的结合位点。除此之外,在提交测序或microarray分析之前,定量PCR是一个快速的方法去测定ChIP样品(提供给我们一些很好的结合位点)。这个方法是为使用罗氏LightCycler而设计的。

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.

材料和试剂

 

1.        PCR 引物

1)         用水从溶于TE 100 μM stocks中准备10 μM引物

2)         选取几个正对照引物和至少一个负对照引物。引物设计原则在这个方法中有。

2.        LightCycler 480 SYBR Green I Master mix (Roche #04 707 516 001)

1)         混合物中含有Taq, dNTPs和嵌入SYBR Green染料。

3.        酵母基因组DNA为正对照稀释级数。

1)         你可以用一个玻璃珠裂解酵母基因组 DNA,它含有RNase处理,Qiagen cleaned并存贮在TE,或者直接从Invitrogen买。

4.        10 μl多孔移液器

1)         带有透明粘合剂塑料盖的384孔反应平板

 

步骤

 

1.        设计PCR引物

Primer3 program (http://frodo.wi.mit.edu/primer3/)可以用于设计引物. 以下引物设计参数对我来说很好用:扩增产物200-250 bp, 退火温度~59-61°C ,引物长度20bp,其他设计参数为Primer3默认参数。

2.        准备一个阳性对照DNA稀释级数

确定扩增效率的每一对引物,准备一个DNA稀释级数。8倍的稀释级数提供一个方便的参考。稀释DNA在纯水中从8倍到87 (2,097,152×)。包括一个没有DNA样品。

3.         在水中稀释ChIP DNA样品

ChIP DNA必须稀释成一个合适的浓度。每一个qPCR反应用2 μl稀释模板

数量是从~4.5 × 109每个ChIP 1/40,它能做10qPCR反应,剩1μl。每个qPCR反应分析的ChIPDNA代表恢复~1 × 107数目。注意:有可能太少的ChIP DNA和数量应该增加两倍。

4.        准备反应混合物(10 μl反应体系)

每个样品准备3次重复(如果数据要发表)或两次重复。我通常准备足够的体积,每8-10反应多一个反应体积,在冰上分所有东西,准备要在黑暗的有盖的容器中进行。

10 μl反应包括如下:

1)         2 μl 纯水

2)         5 μl SYBR Green I Master

3)         1 μl of PCR primers (每个引物0.5μl (从10 μM stock吸取))

4)         2 μl DNA 模板

首先用2 μl纯水稀释SYBR Green I Master mix 每个反应加上5 μl of SYBR Green。为每一对引物分配稀释的SYBR Green I Master mix到想要的体积到管中,加入PCR primers(记得包括额外的反应体积)去准备primer pair master混合物。

5.         建立qPCR反应和加入模板

分配想要的8 μl master反应混合物到384孔反应板的每一个孔中。加入2 μl模板DNA和水到每一个孔中,盖上盖子,去除塑料膜。直到PCR进行前一直保持在黑暗中。

6.        运行qPCR thermocycler程序

除了qPCR样品反应,还应建立每一对引物的溶解曲线分析。

7.         qPCR数据分析

确保每一对引物的溶解曲线是一个单一的峰。通过2-ΔΔCp方法 (2nd derivative maximum) and 计算the mean ΔΔCp value计算两次或三次重复的相对值。对于每一个目标检测,通过负对照计算meanΔΔCp均数的标准差。假定一个正态分布确定数据的统计学意义。

注意/背景

Quantitative PCR (qPCR)用荧光去检测PCR产物的积累。交叉点(Cp)阈值循环(Ct)是当荧光升到略微高于背景的时的点。其他的分析方法用扩增曲线的线性范围。Cp值用于qPCR相对值。qPCR机器检测荧光,软件从荧光的强度计算Cp值。我用扩增曲线的极大二次导数的方法作为标准比较样品。这是一个点,在曲线与最大曲率正向变化。它是信号信号变得能够检测到并进行线性扩增的点。

The SYBR Green方法在PCR反应中用一种可以结合新合成的双链DNA的染料并发出荧光。SYBR Green染料嵌入双链的DNA,使SYBR Green发出荧光。

Real-time PCR也叫做动力PCRqPCR, qRT-PCR and RT-qPCR,是一个定量PCR的方法检测PCR模板的拷贝数,例如DNA或者cDNA。有两种类型的real-time PCR:依赖探针的和依赖嵌入剂的。这两种方法都需要特殊thennocycler,备有敏感的照相机检测在PCR反应频繁间隔的多孔板的每一个孔的荧光值。依赖探针的real-time PCR,也叫做TaqMan PCR,需要像常规PCR一个的引物,一个额外的荧光探针,这个探针是一个含有报告荧光染料和猝灭染料的寡核苷酸。TaqMan方法比SYBR Green方法是更精确可靠,但是更贵些。

 

References

 

1.         Bustin S.A., Benes V., Nolan T., Pfaffl M.W. (2005). Quantitative real-time RT-PCR--a perspective. Journal of Molecular Endocrinology 34(3): 597-601. 

2.         Tichopad A., Dilger M., Schwarz G., Pfaffl M.W. (2003). Standardized determination of real-time PCR efficiency from a single reaction set-up. Nucleic Acids Res 31(20): e122. 

3.         Pfaffl M.W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9): e45. 

 

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How to cite this protocol: Zheng, W. (2011). qPCR of Yeast ChIP DNA. Bio-protocol Bio101: e135. DOI: 10.21769/BioProtoc.135; Full Text



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7/29/2016 8:09:09 AM  

Bioseeker B
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I don't know how to design a primer. Could you please explain about it? Thanks.

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