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Quantitative Methylation Specific PCR (qMSP)
特异检测DNA甲基化修饰的定量PCR(qMSP)   

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Abstract

Detection of low copies of methylated DNA targets in clinical specimens is challenging. The quantitative Methylation-Specific PCR (qMSP) assays were designed to specifically amplify bisulphite-converted methylated DNA target sequences in the presence of an excess of unmethylated counterpart sequences. These qMSP assays are real-time PCR assays utilizing, sequence-specific primers and an intervening, also sequence specific, Taqman probe to cover an amplicon of approximately 100 bp in length. The use of Taqman probes bearing a minor groove binding (MGB) allow for the use of shorter probes and therefore facilitate design and significantly increases the analytical specificity of the reaction. In the context of the biomarker discovery program of the Liverpool Lung Project (LLP), ten gene promoters were selected. qMSP assays were developed, validated and used to screen 655 bronchial washings from patients with lung cancer and age/sex matched controls with non malignant lung disease (Nikolaidis et al., 2012).

Materials and Reagents

  1. TaqMan® Universal Master Mix II no UNG (Life Technologies, catalog number: 4440039 )
  2. TaqMan MGB probes (custom synthesis) (Life Technologies)
  3. EZ DNA Methylation-GoldTM kit (ZymoResearch, catalog number: D5006 )
  4. The methylation-specific primer and probe sequences (see Recipes)

Equipment

  1. PCR cabinet
  2. PCR plates
  3. Centrifuge (Sigma-Aldrich)
  4. PCR thermal cycler (Life Technologies/Applied Biosystems, model: 9700 )
  5. Real time PCR machine (Life Technologies/Applied Biosystems, model: 7500 FAST )

Procedure

  1. Primer/probe design
    Note: Primer/probe design is of major importance for the specificity of the reaction, i.e. to amplify only methylated bisulphite-converted target in the presence of excess unmethylated target. The primers should include 3-5 CG dinucleotides. The inclusion of at least two such CGs within the six 3’ end of the primer significantly increases specificity. This is in addition to the usual rules on primer stability, GC content and secondary structure avoidance apply in real time PCR assay design.
  2. One μg DNA was converted by sodium bisulphite using the EZ DNA Methylation-GoldTM kit and following the supplier’s protocol but eluting in 50 μl elution buffer (instead of the recommended 10 μl).
  3. The qMSP reactions contained 1x TaqMan® Universal Master Mix II non-UNG, 250 nM probe, 300-900 nM primers (Table 1) and 2 μl eluate from the bisulphite treated DNA sample.
    Note: The primer concentration is an important determinant of analytical sensitivity/specificity and has to be ascertained experimentally In other words the analytical sensitivity threshold is set to the dilution that has an overlapping 95% Confidence Interval with the unmethylated control reaction. In practical terms, the highest sensitivity one can use is the one that is always at least 2 ΔCt. lower than the unmethylated control.

    Table 1. Primer-probe concentrations for oligo mixes
    Primer/probe mix
    Final concentration (nM)
    Fwd primer
    Rev Primer
      Probe
    p16
    700
    700
    250
    TERT
    250
    250
    250
    RASSF1
    700
    700
    250
    TMEFF2
    900
    900
    250
    CYGB
    300
    300
    250
    RARb
    500
    500
    250
    DAPK1
    250
    250
    250
    p73
    250
    250
    250
    WT1
    750
    750
    250
    CDH13
    250
    250
    250
    ACTB
    900
    900
    250

  4. PCR plates were sealed and span at 4,000 x g for 1 min prior to be placed in the thermal cycler in order to bring all the reaction volume to the bottom and ensure removal of bubbles from the reaction mix.
  5. The reactions were performed in duplicate on a 7500 FAST real time cycler under the following thermal profile: 95 °C for 10 min activation step followed by 50 cycles consisted of denaturation at 95 °C for 15 sec, annealing and extension at 58 °C–65 °C (Table 2, depending on the assay) for 1 min.

    Table 2. Annealing information for qMSP optimised conditions
    Genes
    Annealing temp (°C)
    Time (sec)
    p16
    60
    60
    RASSF1
    60
    60
    CYGB
    64
    5
    61
    55
    RARB
    65
    5
    62
    55
    TERT
    65
    5
    62.5
    55
    WT
    62
    60
    ACTB
    58
    20
    60
    40
    CDH13
    64
    5
    61
    55
    DAPK
    65
    5
    62.5
    55
    P73
    65
    5
    62.5
    55
    TMEFF
    58
    20
    60
    40

Recipes

  1. The methylation-specific primer and probe sequences are listed in Table 3. In the initial steps of assay development it became apparent that probes bearing minor groove binding moiety (Taqman MGB probes) provided significantly higher assay specificity. In addition, due to their smaller size, they allow for a more flexible assay design.

    Table 3. Nucleotide sequences of methylation specific primers and probes for the qMSP assays utilised in the BW screening. The ACTB assay is methylation-independent acting as DNA input control.
    Primer/probe name
    Sequence 5’ →3’
    Modification
    p16meth_F
    GGAGGGGGTTTTTTCGTTAGTATC

    p16meth_R
    CTACCTACTCTCCCCCTCTCCG

    p16meth_P
    AACGCACGCGATCC
    FAM-MGB
    RASSF1meth_F
    GTGGTGTTTTGCGGTCGTC

    RASSF1meth_R
    AACTAAACGCGCTCTCGCA

    RASSF1_P
    CGTTGTGGTCGTTCG
    FAM-MGB
    TMEFF2meth_F
    GGAGAGTTAAGGCGTTTCGTAGTTC

    TMEFF2meth_R
    CGTGGGAAGAGGTAGTCGGG

    TMEFF2meth_P
    GTTTTTAGTTCGTTCG
    FAM-MGB
    TERTmeth_F
    TTGGGAGTTCGGTTTGGTTTC

    TERTmeth_R
    CACCCTAAAAACGCGAACGA

    TERTmeth_P
    AGCGTAGTTGTTTCGG
    FAM-MGB
    CYGBmeth_F
    GTGTAATTTCGTCGTGGTTTGC

    CYGBmeth_R
    CCGACAAAATAAAAACTACGCG

    CYGBmeth_P
    TGGGCGGGCGGTAG
    FAM-MGB
    RARbmeth_F
    GATTGGGATGTCGAGAACGC

    RARbmeth_R
    ACTTACAAAAAACCTTCCGAATACG

    RARbmeth_P
    AGCGATTCGAGTAGGGT
    FAM-MGB
    DAPK1meth_F
    CGAGCGTCGCGTAGAATTC

    DAPK1meth_R
    ACCCTACAAACGAACTAACGACG

    DAPK1meth_P
    AGCGTCGGTTTGGTAG
    FAM-MGB
    p73meth_F
    TTGTTTTTTGGATTTTAAGCGTTTC

    p73meth_R
    CACCCGAATCTCTCCTAACCG

    p73meth_P
    TAACGCTAAACTCCTCG
    FAM-MGB
    WT1meth_F
    GAGGAGTTAGGAGGTTCGGTC

    WT1meth_R
    CACCCCAACTACGAAAACG

    WT1meth_P
    AGTTCGGTTAGGTAGC
    FAM-MGB
    CDH13meth_F
    CGTGTATGAATGAAAACGTCGTC

    CDH13meth_R
    CACAAAACGAACGAAATTCTCG

    CDH13meth_P
    CGTTTTTAGTCGGATAAAA
    FAM-MGB
    ACTBmgb_F
    GGGTGGTGATGGAGGAGGTT

    ACTBmgb_R
    TAACCACCACCCAACACACAAT

    ACTBmgb_P
    TGGATTGTGAATTTGTGTTTG
    VIC-MGB

    Cycle threshold (Ct) values for each target were normalized for DNA input by calculating the ΔCt=Ct(Target)-Ct(ACTB). The values for al samples were transformed to relative quantity (RQ) compare to the calibrator (0.5% standard methylated DNA dilution) included in all experiments using the following type:
    RQsample = 2-ΔΔCt, whereΔΔCt = ΔCtsample - ΔCtcalibrator.
    Note: qMSP is a challenging version of real-time PCR and one needs to gain a very good understanding of the latter prior to engaging in qMSP experiments. The main additional challenge is the use of bisulphite DNA which is of lower quality but most importantly of lower complexity. This significantly affects the thermodynamic behavior of this template in the reaction. The authors are very happy to provide assistance to colleagues if needed; please email Dr. T Liloglou (tliloglo@liv.ac.uk).

Acknowledgments

This protocol is adapted from Nikolaidis et al. (2012).

References

  1. Liloglou, T., Bediaga, N. G., Brown, B. R., Field, J. K. and Davies, M. P. (2012). Epigenetic biomarkers in lung cancer. Cancer Lett 342(2): 200-212.
  2. Nikolaidis, G., Raji, O. Y., Markopoulou, S., Gosney, J. R., Bryan, J., Warburton, C., Walshaw, M., Sheard, J., Field, J. K. and Liloglou, T. (2012). DNA methylation biomarkers offer improved diagnostic efficiency in lung cancer. Cancer Res 72(22): 5692-5701.

简介

在临床标本中检测低拷贝的甲基化DNA靶是具有挑战性的。设计定量甲基化特异性PCR(qMSP)测定以在过量的未甲基化的对应物序列存在下特异性扩增亚硫酸氢盐转化的甲基化DNA靶序列。这些qMSP测定法是实时PCR测定,利用序列特异性引物和间插的,也是序列特异性的Taqman探针以覆盖长度约100bp的扩增子。使用具有小沟结合(MGB)的Taqman探针允许使用更短的探针,因此便于设计并显着增加反应的分析特异性。在利物浦肺项目(LLP)的生物标志物发现程序的上下文中,选择十个基因启动子。 qMSP测定法被开发,验证并用于筛选来自患有肺癌和具有非恶性肺疾病的年龄/性别匹配对照的患者的支气管洗液655(Nikolaidis等人,2012)。

材料和试剂

  1. TaqMan ® Universal Master Mix II no UNG(Life Technologies,catalogue number:4440039)
  2. TaqMan MGB探针(定制合成)(Life Technologies)
  3. EZ DNA甲基化-Gold TM 试剂盒(ZymoResearch,目录号:D5006)
  4. 甲基化特异性引物和探针序列(参见配方)

设备

  1. PCR室
  2. PCR板
  3. 离心机(Sigma-Aldrich)
  4. PCR热循环仪(Life Technologies/Applied Biosystems,型号:9700)
  5. 实时PCR仪(Life Technologies/Applied Biosystems,型号:7500FAST)

程序

  1. 底漆/探针设计
    注意:引物/探针设计对于反应的特异性是非常重要的,即在过量的未甲基化的靶的存在下扩增仅甲基化的亚硫酸氢盐转化的靶。引物应包括3-5个CG二核苷酸。在引物的6个3'末端内包含至少两个这样的CG显着增加了特异性。这是除了引物稳定性,GC含量和二级结构避免的通常规则适用于实时PCR测定设计。
  2. 使用EZ DNA甲基化-Gold< sup>试剂盒并根据供应商的方案,但是在50μl洗脱缓冲液(而不是推荐的10μl)中洗脱,通过亚硫酸氢钠将1μgDNA转化。
  3. qMSP反应包含1×TaqMan通用Master Mix II非UNG,250nM探针,300-900nM引物(表1)和2μl来自亚硫酸氢盐处理的DNA样品的洗脱液。
    注意:引物浓度是分析灵敏度/特异性的重要决定因素,并且必须通过实验确定。换句话说,分析灵敏度阈值设置为与未甲基化对照反应具有重叠95%置信区间的稀释度。实际上,可以使用的最高灵敏度是总是至少为2ΔCt的灵敏度。低于未甲基化对照。

    表1.寡核苷酸混合物的引物探针浓度
    引物/探针混合物
    最终浓度(nM)
    前导
    Rev Primer
      探头
    p16
    700
    700
    250
    TERT
    250
    250
    250
    RASSF1
    700
    700
    250
    TMEFF2
    900
    900
    250
    CYGB
    300
    300
    250
    RARb
    500
    500
    250
    DAPK1
    250
    250
    250
    p73
    250
    250
    250
    WT1
    750
    750
    250
    CDH13
    250
    250
    250
    ACTB
    900
    900
    250

  4. 将PCR板密封并以4,000×g跨越1分钟,然后置于热循环仪中,以使所有反应体积到底部,并确保从反应混合物中除去气泡。 />
  5. 在7500FAST实时循环仪上,在以下热曲线下进行一式两份的反应:95℃10分钟活化步骤,然后是50个循环,包括95℃变性15秒,退火和58℃-100℃延伸, 65℃(表2,取决于测定)1分钟
    表2. qMSP优化条件的退火信息
    基因
    退火温度(℃)
    时间(秒)
    p16
    60
    60
    RASSF1
    60
    60
    CYGB
    64
    5
    61
    55
    RARB
    65
    5
    62
    55
    TERT
    65
    5
    62.5
    55
    WT
    62
    60
    ACTB
    58
    20
    60
    40
    CDH13
    64
    5
    61
    55
    DAPK
    65
    5
    62.5
    55
    P73
    65
    5
    62.5
    55
    TMEFF
    58
    20
    60
    40

食谱

  1. 甲基化特异性引物和探针序列列于表3中。在测定开发的初始步骤中,显而易见的是,具有小沟结合部分(Taqman MGB探针)的探针提供了显着更高的测定特异性。 此外,由于其尺寸较小,它们允许更灵活的测定设计
    表3.甲基化特异性引物和探针的核苷酸序列 用于BW筛选中使用的qMSP测定。 ACTB测定 甲基化独立作为DNA输入控制
    引物/探针名称
    顺序5'→3'
    修改
    p16meth_F
    GGAGGGGGTTTTTTCGTTAGTATC

    p16meth_R
    CTACCTACTCTCCCCCTCTCCG

    p16meth_P
    AACGCACGCGATCC
    FAM-MGB
    RASSF1meth_F
    GTGGTGTTTTGCGGTCGTC

    RASSF1meth_R
    AACTAAACGCGCTCTCGCA

    RASSF1_P
    CGTTGTGGTCGTTCG
    FAM-MGB
    TMEFF2meth_F
    GGAGAGTTAAGGCGTTTCGTAGTTC

    TMEFF2meth_R
    CGTGGGAAGAGGTAGTCGGG

    TMEFF2meth_P
    GTTTTTAGTTCGTTCG
    FAM-MGB
    TERTmeth_F
    TTGGGAGTTCGGTTTGGTTTC

    TERTmeth_R
    CACCCTAAAAACGCGAACGA

    TERTmeth_P
    AGCGTAGTTGTTTCGG
    FAM-MGB
    CYGBmeth_F
    GTGTAATTTCGTCGTGGTTTGC

    CYGBmeth_R
    CCGACAAAATAAAAACTACGCG

    CYGBmeth_P
    TGGGCGGGCGGTAG
    FAM-MGB
    RARbmeth_F
    GATTGGGATGTCGAGAACGC

    RARbmeth_R
    ACTTACAAAAAACCTTCCGAATACG

    RARbmeth_P
    AGCGATTCGAGTAGGGT
    FAM-MGB
    DAPK1meth_F
    CGAGCGTCGCGTAGAATTC

    DAPK1meth_R
    ACCCTACAAACGAACTAACGACG

    DAPK1meth_P
    AGCGTCGGTTTGGTAG
    FAM-MGB
    p73meth_F
    TTGTTTTTTGGATTTTAAGCGTTTC

    p73meth_R
    CACCCGAATCTCTCCTAACCG

    p73meth_P
    TAACGCTAAACTCCTCG
    FAM-MGB
    WT1meth_F
    GAGGAGTTAGGAGGTTCGGTC

    WT1meth_R
    CACCCCAACTACGAAAACG

    WT1meth_P
    AGTTCGGTTAGGTAGC
    FAM-MGB
    CDH13meth_F
    CGTGTATGAATGAAAACGTCGTC

    CDH13meth_R
    CACAAAACGAACGAAATTCTCG

    CDH13meth_P
    CGTTTTTAGTCGGATAAAA
    FAM-MGB
    ACTBmgb_F
    GGGTGGTGATGGAGGAGGTT

    ACTBmgb_R
    TAACCACCACCCAACACACAAT

    ACTBmgb_P
    TGGATTGTGAATTTGTGTTTG
    VIC-MGB

    通过计算ΔCt= Ct(靶)-Ct(ACTB)来对于每个靶的循环阈值(Ct)值进行标准化。与包括在使用以下类型的所有实验中的校准物(0.5%标准甲基化DNA稀释物)相比,将α样品的值转化为相对量(RQ):
    RQ子样品 = 2 -ΔΔCt,其中ΔΔCt=ΔCt样品 注意:qMSP是实时PCR的具有挑战性的版本,需要在进行qMSP实验之前获得对后者的非常好的理解。主要的额外挑战是使用质量较低但最重要的是较低复杂性的亚硫酸氢盐DNA。这显着影响该模板在反应中的热力学行为。作者非常乐意在需要时向同事提供帮助;请发送电子邮件至Dr. T Liloglou( tliloglo@liv.ac.uk )。

致谢

该协议改编自Nikolaidis等人(2012)。

参考文献

  1. Liloglou,T.,Bediaga,N.G.,Brown,B.R.,Field,J.K.and Davies,M.P。(2012)。 肺癌中的表观遗传学生物标志物。 Cancer Lett 342(2):200-212。
  2. Nikolaidis,G.,Raji,O.Y.,Markopoulou,S.,Gosney,J.R.,Bryan,J.,Warburton,C.,Walshaw,M.,Sheard,J.,Field,J.K.and Liloglou, DNA甲基化生物标志物可提高肺癌的诊断效率。 Cancer Res 72(22):5692-5701。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liloglou, T. and Nikolaidis, G. (2013). Quantitative Methylation Specific PCR (qMSP). Bio-protocol 3(16): e871. DOI: 10.21769/BioProtoc.871.
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