DNA Methylation Profiling Using Infinium Methylation Assay
采用Infinium 甲基化试验进行DNA甲基化分析   

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Oct 2012



The Infinium Human Methylation 450 BeadChip technology allows the rapid quantitative DNA methylation analysis of more than 485,000 CpG dinucleotides located across the genome. The method utilizes sodium bisulfite treatment of genomic DNA to convert unmethylated cytosine residues into uracils whereas methylated cytosines remain unchanged. Modified DNA is then whole genome amplified, fragmented and hybridized to locus-specific oligomer probes linked to individual beads on a BeadChip. Hybridization is followed by single-base extension of the oligomer with a labeled nucleotide. The BeadChip is subsequently fluorescently stained and scanned to measure the intensities of the beads corresponding to the unmethylated and methylated CpG sites.

Keywords: Epigenetics (epigenetics), DNA methylation (DNA甲基化), Infinium Methylation Assay (infinium甲基化检测), Bisulfite conversion (亚硫酸氢钠的转换), Cancer Biology (癌症生物学)

Materials and Reagents

  1. EZ-96 DNA Methylation Kit (Zymo Research, catalog number: D5003 )
  2. Ethanol (VWR International, catalog number: 20821.330 )
  3. Genomic DNA
  4. PCR Grade water (Life Technologies, InvitrogenTM, catalog number: AM9937 )
  5. PCR reagents: PCR buffer, dNTPs, Taq (Roche Applied Science, catalog number: 04738420001 )
  6. Primers for methylation specific PCR [see Step 20-(1)]
  7. 96-well 0.2 ml microplates (Starlab, catalog number: I1402-9800-C )
  8. 96-well 0.8 ml microplates (Thermo Fisher Scientific, catalog number: AB-0765 )
  9. Seal sheets (Thermo Fisher Scientific, catalog number: AB-0558 )
  10. Infinium Human Methylation450 BeadChip Kit (Illumina, catalog number: WG-314-1003 )
  11. PyroMark Q96 CpG LINE-1 Kit (QIAGEN, catalog number: 973043 ) and associated reagents (Optional)


  1. 96-well Thermal Cycler
  2. Centrifuge with microplate adapters
  3. Vortex mixer
  4. HiScan SQ or iScan systems (Illumina, catalog number: SY-103-2001 or SY-101-1001 respectively)
  5. GenomeStudio Methylation Module software (Illumina, catalog number: SW-300-1001 )
  6. NanoDrop ND-1000 UV-Vis spectrophotometer (Thermo Fisher Scientific) or equivalent
  7. Speed Vacuum (Optional)
  8. PyroMark Q96 MD (QIAGEN, catalog number: 9001526 ) or equivalent (Optional)


  1. Quantitate genomic DNA concentration using NanoDrop and determine A260/280 ratio which should be > 1.80 for samples to be analyzed.
  2. Dilute each DNA sample in water to 100 ng μl-1 concentration.
    Note: It is also possible to use samples at lower concentrations and adjust the volumes in step 6 accordingly to meet the minimum quantity of DNA required. However, volumes of DNA samples with lower concentrations used in step 6 should not exceed a maximum volume of 45 μl.
    Note: Once DNA is bisulfite treated it should be used for Infinium Methylation analyses as soon as possible, preferably within one month.
  3. Prepare Conversion Reagent from EZ96 DNA Methylation Kit by adding 7.5 ml water and 2.1 ml of M-Dilution buffer to a bottle of CT Conversion Reagent. Mix at room temperature with frequent vortexing for 10 min and keep reagent in dark.
    Note: It is normal to see trace amounts of undissolved CT Conversion Reagent. Also, for best results the reagent should be immediately used after preparation.
  4. Prepare M-Wash Buffer from EZ96 DNA Methylation Kit by adding 144 ml 100% ethanol to the 36 ml M-Wash Buffer concentrate.
  5. Pipet 10 μl of each DNA sample (100 ng μl-1) into a 96-well 0.2 ml microplate for bisulfite treatment (500 ng is the minimum quantity of DNA used).
  6. Add 5 μl of M-Dilution buffer to each sample and adjust the total volume to 50 μl with water and mix by pipetting up and down. Seal with seal sheet.
  7. Incubate samples at 37 °C for 15 min using thermal cycler.
  8. Add 100 μl of the prepared CT Conversion Reagent to each sample and mix by pipetting up and down several times. Seal plate with a seal sheet.
    Note: CT-Conversion Reagent contains 97% Sodium Metabisulfite (bisulfite ions) which mediates bisulfite-mediated deamination of unmethylated Cytosine.
  9. Incubate samples on thermal cycler using the following program
    1. 95 °C for 30 sec.
    2. 50 °C for 1 h and repeat steps a and b for 15 more cycles.
    3. 4 °C for 10 min.
  10. From EZ96 DNA Methylation Kit, place Silicon-A Binding Plate on top of Collection Plate and pipette 400 μl of M-Binding Buffer to each well. This buffer contains guanidine hydrochloride salt which provides pH that enables DNA to bind to the silica particles and allows washing and bisulfite treatment of the DNA.
  11. Load the DNA samples from step 10 into the wells of the Silicon-A Binding Plate and mix by pipetting up and down.
  12. Centrifuge at room temperature at ≥ 3,000 x g for 5 min and discard the flow-through. Use this condition for all centrifugation steps in this protocol.
  13. Add 500 μl of M-Wash buffer to each well and centrifuge at ≥ 3,000 x g for 5 min. Discard the flow-through. M-Wash buffer is to wash and clean any reagent from silica-bound DNA from previous step.
  14. Add 200 μl of M-Desulphonation buffer to each well and let it stand at room temperature for 15-20 min. After incubation centrifuge at ≥ 3,000 x g for 5 min. Discard the flow-through. M-Desulphonation buffer contains sodium hydroxide which converts deaminated cytosines to Uracil.
  15. Add 500 μl of M-Wash buffer to each well and centrifuge at ≥ 3,000 x g for 5 min. Discard the flow-through.
  16. Add another 500 μl of M-Wash buffer to each well and centrifuge at ≥ 3,000 x g for 10 min. Ensure there is no liquid left in the wells of Silicon-A binding Plate, otherwise centrifuge for further 2 min.
  17. Place Silicon A Binding Plate onto an elution plate.
  18. Add 30 μl of M-Elution buffer directly to the binding matrix in each well of Silicon A Binding Plate. Incubate for 1-2 min at room temperature and centrifuge for 3 min at ≥ 3,000 x g to elute the DNA. M-Elution buffer contains 1% Tris (hydroxymethyl) aminomethane/Hydrochloric Acid and 1% Ethylenediaminetetraacetic Acid, pH 8.0 which enables dissociation of DNA from silica column.
  19. Keep aside 4 μl of each (bisulfite converted) DNA sample for quality control and store remaining amount in the elution plate at -20 °C.
    Note: It is recommended to use bisulfite converted samples on the Infinium HumanMethylation450 BeadChip within 1 month of conversion and to avoid repeated freeze-thaw cycles of samples.
  20. Assess the quality of bisulfite converted DNA by using for instance methylation-specific PCR (Ku et al., 2011) for gene of interest (see Note below for an example of our Methylation-specific PCR for Calponin gene) or using pyrosequencing of LINE1 with PyroMark Q96 CpG LINE-1 kit. Proceed with DNA samples which have passed quality control, i.e. samples which either show a PCR product with methylation specific PCR or have passed the bisulfite modification control contained within the LINE1 pyrosequencing assay.
    1. Methylation-specific PCR for Calponin gene for assessing quality of bisulfite treated DNA:
      Mix 15.8 μl water with
      2.5 μl 10x PCR buffer
      3 μl MgCl2 (25 mM)
      0.5 μl dNTPs (2.5 mM)
      1 μl Forward Primer (5′-GGAAGGTAGTTGAGGTTGTG-3′, 20 μM)
      1 μl Reverse Primer (5′-CCCAAACTCAAAACTCTAACCTAAC-3′, 20 μM)
      1 μl Bisulfite converted DNA
      0.2 μl Taq (5 U)
    2. PCR conditions:
      95 °C for 6 min
      95 °C for 30 sec
      63 °C for 30 sec
      72 °C for 30 sec
      Repeat steps b to d for 35 cycles
      72 °C for 5 min.
      Run PCR products on a 2% agarose gel to confirm presence of amplicons (333 bp) which verify that the bisulfite conversion has been successful in respective samples. (Teodoridis et al., 2005)

  21.  Determine concentration of bisulfite treated DNA using NanoDrop.
    Note: As the bisulfite treatment can degrade DNA samples, we have additional DNA quantitation step here after the bisulfite treatment in our protocol, to ensure that there is sufficient sample present before proceeding to Infinium Methylation analyses.
  22. Thaw DNA samples stored at -20 °C and briefly centrifuge. Adjust DNA concentration of each sample to 50 ng μl-1 with M-Elution buffer or water.
    Note: DNA samples below 50 ng μl -1 may be concentrated using speed vacuum.
  23. Transfer 6 μl of each 50 ng μl-1 sample to a new 0.8 ml 96-well microplate which is labeled as BCD plate in the Infinium HD Assay Methylation Protocol.
  24. Process 4 μl (or equivalent of 200 ng) samples according to Infinium HD Assay Methylation Protocol Guide (see Reference 3), starting from step 3, page 43 (under steps to make MSA4 plate). Briefly, bisulfite converted DNA is whole genome amplified, fragmented and hybridized to the BeadChip. Following hybridization, unhybridized DNA is washed off and probes on the BeadChip are extended using a labeled nucleotide and captured DNA as a template. The BeadChip is fluorescently stained, scanned and the intensities of beads are measured.
  25. Following processing, extract DNA methylation signals from scanned arrays.
  26. Background correct raw signals and compute into β value using the GenomeStudio software.
  27. Before proceeding to data analysis, perform quality control using the GenomeStudio Software. Data quality can be assessed by checking the detection p-value of probes as well as the sample-dependent and sample-independent control probes which are present on the BeadChips. The following criteria are applied for removal of poorly performing probes or samples: a) probes with a detection p-value above a certain cut-off (e.g. p < 0.05) should be excluded from the analysis, b) samples which fail in any of the array control probes i.e. the raw signal intensities of the control probes of the sample are out of normal range (median+3SD or median-3SD depending on the control probe type) of the signal intensities across all the samples and c) if a certain probe has failed quality control via the detection p-value in greater than 10-25% of samples, then that probe should be removed from further downstream analyses.
  28. An appropriate method of within and between array normalization should be performed. For instance the Genome studio software provides an internal control normalization procedure.
  29. The Infinium 450 K BeadChips use two different types of assay chemistries (Infinium I and Infinium II probes). The β value represents either the ratio of the intensity of the methylated bead type to the combined locus intensity (Infinium type 1 probes) or the ratio of the intensity of the signal from the green channel (corresponding to the methylated CpG state) to the combined intensity obtained from red and green channels (Infinium type II probes) and reflects the methylation status of the specific CpG site. Due to the divergent chemistries of Infinium I and II probes which result in different β value distributions it is recommended to perform peak base correction or the scaling of probes to more accurately reflect the divergent distributions of type I and II probes. Different peak base correction methods are publicly available; see for instance in (Teschendorff et al., 2013).


This work was supported by grant awards from Cancer Research UK (C536/A6689), Imperial Experimental Cancer Medicine Centre and Imperial Cancer Research UK Centre.


  1. Dedeurwaerder, S., Defrance, M., Calonne, E., Denis, H., Sotiriou, C. and Fuks, F. (2011). Evaluation of the Infinium Methylation 450K technology. Epigenomics 3(6): 771-784. 
  2. EZ-96 DNA Methylation Kit D5003 Instruction Manual Version 1.2.6, Zymo Research.
  3. GenomeStudio Methylation Module v1.8 User Guide, Catalog number # 11319130 Rev. B November 2010, Illumina. 
  4. Infinium HD Assay Methylation Protocol Guide, catalog number: WG-914-1001Pat # 15019519 Rev. A December 2010, Illumina.
  5. Ku, J. L., Jeon, Y. K., Park, J. G. (2011). Methylation-specific PCR. Methods Mol Biol 791:23-32. 
  6. PyroMark Q96 CpG LINE-1 Handbook 12/2010, Qiagen.
  7. Sriraksa, R., Chaopatchayakul, P., Jearanaikoon, P., Leelayuwat, C. and Limpaiboon, T. (2010). Verification of complete bisulfite modification using Calponin-specific primer sets. Clin Biochem 43(4-5): 528-530. 
  8. Teschendorff, A. E., Marabita, F., Lechner, M., Bartlett, T., Tegner, J., Gomez-Cabrero, D., Beck, S. (2013). A Beta-Mixture Quantile Normalisation method for correcting probe design bias in Illumina infinium 450k DNA methylation data. Bioinformatics 29 (2): 189-196.
  9. Teodoridis, J. M., Hall, J., Marsh, S., Kannall, H. D., Smyth, C., Curto, J., Siddiqui, N., Gabra, H., McLeod, H. L., Strathdee, G. and Brown, R. (2005). CpG island methylation of DNA damage response genes in advanced ovarian cancer. Cancer Res 65(19): 8961-8967. 
  10. Touleimat, N. and Tost, J. (2012). Complete pipeline for Infinium((R)) Human Methylation 450K BeadChip data processing using subset quantile normalization for accurate DNA methylation estimation. Epigenomics 4(3): 325-341. 
  11. Zeller, C., Dai, W., Steele, N. L., Siddiq, A., Walley, A. J., Wilhelm-Benartzi, C. S., Rizzo, S., van der Zee, A., Plumb, J. A. and Brown, R. (2012). Candidate DNA methylation drivers of acquired cisplatin resistance in ovarian cancer identified by methylome and expression profiling. Oncogene 31(42): 4567-4576. 


Infinium人甲基化450 BeadChip技术允许对跨越基因组的超过485,000个CpG二核苷酸进行快速定量DNA甲基化分析。 该方法利用亚硫酸氢钠处理基因组DNA以将未甲基化的胞嘧啶残基转化为尿嘧啶,而甲基化的胞嘧啶保持不变。 然后将修饰的DNA全基因组扩增,片段化并与连接到BeadChip上的单个珠的基因座特异性寡核苷酸探针杂交。 杂交之后是具有标记的核苷酸的寡聚物的单碱基延伸。 随后对珠粒芯片进行荧光染色和扫描,以测量对应于未甲基化和甲基化的CpG位点的珠子的强度。

关键字:epigenetics, DNA甲基化, infinium甲基化检测, 亚硫酸氢钠的转换, 癌症生物学


  1. EZ-96 DNA甲基化试剂盒(Zymo Research,目录号:D5003)
  2. 乙醇(VWR International,目录号:20821.330)
  3. 基因组DNA
  4. PCR级水(Life Technologies,Invitrogen TM ,目录号:AM9937)
  5. PCR试剂:PCR缓冲液,dNTPs,Taq(Roche Applied Science,目录号:04738420001)
  6. 用于甲基化特异性PCR的引物[参见步骤20-(1)]
  7. 96孔0.2ml微量培养板(Starlab,目录号:I1402-9800-C)
  8. 96孔0.8ml微量培养板(Thermo Fisher Scientific,目录号:AB-0765)
  9. 密封片(Thermo Fisher Scientific,目录号:AB-0558)
  10. Infinium Human Methylation450 BeadChip Kit(Illumina,目录号:WG-314-1003)
  11. PyroMark Q96 CpG LINE-1试剂盒(QIAGEN,目录号:973043)和相关试剂(可选)


  1. 96孔热循环仪
  2. 用微孔板适配器离心
  3. 涡流搅拌器
  4. HiScan SQ或iScan系统(Illumina,目录号:SY-103-2001或SY-101-1001)
  5. GenomeStudio甲基化模块软件(Illumina,目录号:SW-300-1001)
  6. NanoDrop ND-1000紫外 - 可见分光光度计(Thermo Fisher Scientific)或等同物
  7. 速度真空(可选)
  8. PyroMark Q96 MD(QIAGEN,目录号:9001526)或等同物(可选)


  1. 使用NanoDrop定量基因组DNA浓度并确定A 260/280比率,其应该> 1.80用于待分析的样品。
  2. 将每个DNA样品在水中稀释至100ng /μl浓度。
  3. 通过向一瓶CT转化试剂中加入7.5 ml水和2.1 ml M-Dilution缓冲液,从EZ96 DNA甲基化试剂盒制备转化试剂。在室温下混合,经常涡旋10分钟,并保持试剂在黑暗中 注意:看到痕量的未溶解的CT转换试剂是正常的。此外,为了获得最佳效果,试剂应在制备后立即使用。
  4. 通过加入144 ml 100%乙醇到36 ml M-Wash Buffer浓缩液中,从EZ96 DNA甲基化试剂盒制备M-Wash缓冲液。
  5. 吸取10μl的每个DNA样品(100ng /μl)到用于亚硫酸氢盐处理的96孔0.2ml微量培养板中(500ng是所使用的DNA的最小量)。
  6. 向每个样品中加入5μlM-Dilution缓冲液,用水调整总体积至50μl,并通过上下吹吸混匀。用密封片密封。
  7. 使用热循环仪在37℃孵育样品15分钟
  8. 向每个样品中加入100μl制备的CT转化试剂,并通过上下吹吸数次混合。 密封板带密封片。
  9. 使用以下程序在热循环仪上孵育样品
    1. 95℃30秒。
    2. 50℃下1小时,并重复步骤a和b再进行15个循环。
    3. 4℃10分钟。
  10. 从EZ96 DNA甲基化试剂盒,将硅A结合板放置在收集板的顶部,并移取400μlM-结合缓冲液到每个孔中。该缓冲液含有胍盐酸盐,其提供使DNA能够与二氧化硅颗粒结合并允许洗涤和亚硫酸氢盐处理DNA的pH。
  11. 将步骤10的DNA样品装入硅胶结合板的孔中,通过上下吹吸混合。
  12. 在室温下以≥3,000xg离心5分钟,弃去流出液。在此协议中的所有离心步骤中使用此条件。
  13. 向每个孔中加入500μlM-Wash缓冲液,并在≥3,000xg离心5分钟。丢弃流通。 M-洗涤缓冲液是从前面步骤中的二氧化硅结合的DNA洗涤和清洁任何试剂。
  14. 向每个孔中加入200μl的M-脱磺化缓冲液,并使其在室温下静置15-20分钟。在孵育后在≥3,000xg下离心5分钟。丢弃流通。 M-脱磺化缓冲液含有将脱氨胞嘧啶转化成尿嘧啶的氢氧化钠。
  15. 向每个孔中加入500μlM-Wash缓冲液,并在≥3,000xg离心5分钟。丢弃流出物。
  16. 向每个孔中再加入500μlM-Wash缓冲液,并在≥3,000xg离心10分钟。确保硅-A结合板的孔中没有液体,否则离心2分钟
  17. 将硅A结合板放在洗脱板上。
  18. 在硅A结合板的每个孔中向结合基质直接加入30μlM-洗脱缓冲液。在室温下孵育1-2分钟,在≥3,000xg离心3分钟以洗脱DNA。 M-洗脱缓冲液含有1%Tris(羟甲基)氨基甲烷/盐酸和1%乙二胺四乙酸,pH 8.0,使得DNA能够从二氧化硅柱上解离。
  19. 保留4μl每个(亚硫酸氢盐转换的)DNA样品用于质量控制,并在-20℃下将剩余量存储在洗脱板中。
    注意:建议在转化后1个月内,在Infinium HumanMethylation450 BeadChip上使用经亚硫酸氢盐转换的样品,并避免样品反复冻融。
  20. 通过使用例如甲基化特异性PCR(Ku等人,2011)针对感兴趣的基因来评估亚硫酸氢盐转化的DNA的质量(参见下面关于我们的用于Calponin基因的甲基化特异性PCR的实例)或使用PyroMark Q96 CpG LINE-1试剂盒对LINE1进行焦磷酸测序。继续通过质量控制的DNA样品,即显示具有甲基化特异性PCR的PCR产物或通过亚硫酸氢盐的样品 包含在LINE1焦磷酸测序测定中。
    混合     2.5μl      3μlMgCl 2(25mM)
        0.5μldNTP(2.5 mM)
        0.2μlTaq(5 U)
    2) PCR条件:
        a。  95℃6分钟
        b。  95℃30秒
        c。  63°C 30秒
        d。 72°C 30秒
       重复步骤b到d 35个周期
    在2%琼脂糖凝胶上运行PCR产物以确认扩增子(333bp)的存在,其证实亚硫酸氢盐转化在各样品中成功。 (Teodoridis et al。,2005)
  21.  使用NanoDrop确定亚硫酸氢盐处理的DNA的浓度。
  22. 解冻DNA样品储存在-20°C,并短暂离心。用M-洗脱缓冲液或水将每个样品的DNA浓度调节至50ng /μl。
    注意:低于50 ngμl -1 的DNA样本可能会使用真空浓缩。
  23. 在Infinium HD Assay Methylation Protocol中将6μl的每个50 ngμl -1样品转移到一个新的0.8 ml 96孔微孔板中,该微孔板标记为BCD板。
  24. 根据Infinium HD Assay Methylation Protocol Guide(参见参考文献3),从第43页的步骤3(在制备MSA4板的步骤)开始,处理4μl(或等价的200ng)样品。简言之,将亚硫酸氢盐转化的DNA全基因组扩增,片段化并与BeadChip杂交。杂交后,洗掉未杂交的DNA,使用标记的核苷酸和捕获的DNA作为模板扩增BeadChip上的探针。将BeadChip荧光染色,扫描并测量珠的强度。
  25. 在处理后,从扫描阵列提取DNA甲基化信号。
  26. 背景校正原始信号并使用GenomeStudio软件计算为β值
  27. 在进行数据分析之前,请使用GenomeStudio软件进行质量控制。可以通过检查探针的检测p值以及存在于珠粒芯片上的样品依赖性和样品非依赖性对照探针来评估数据质量。将以下标准应用于去除效能差的探针或样品:a)应该从分析中排除检测p值高于某一截止值(例如p <0.05)的探针, b)任何阵列控制探针中出现故障的样品,即样品的对照探针的原始信号强度超出所有样品的信号强度的正常范围(中位数+ 3SD或中位-3SD,取决于对照探针类型)样品和c)如果某个探针通过检测p值在大于10-25%的样品中的质量控制失败,那么该探针应当从进一步的下游分析中去除。
  28. 应该执行在阵列标准化之内和之间的适当方法。例如,Genome工作室软件提供内部对照归一化程序。
  29. Infinium 450 K BeadChip使用两种不同类型的测定化学成分(Infinium I和Infinium II探针)。 β值表示甲基化珠类型的强度与组合的基因座强度(Infinium 1型探针)的比率或来自绿色通道(对应于甲基化的CpG态)的信号的强度与组合的从红色和绿色通道获得的强度(Infinium II型探针) 并反映特定CpG位点的甲基化状态。由于导致不同β值分布的Infinium I和II探针的不同化学性质,推荐进行峰值碱基校正或探针的标度以更准确地反映I型和II型探针的发散分布。不同的峰基校正方法是公众可获得的;参见例如(Teschendorff等人,2013)。




  1. Dedeurwaerder,S.,Defrance,M.,Calonne,E.,Denis,H.,Sotiriou,C.and Fuks,F。(2011)。 评估Infinium甲基化450K技术。 3(6):771-784。
  2. EZ-96 DNA甲基化试剂盒D5003使用说明书版本1.2.6,Zymo研究
  3. GenomeStudio Methylation Module v1.8用户指南,目录号#11319130 Rev. B 2010年11月,Illumina。
  4. Infinium HD Assay Methylation Protocol Guide,目录号:WG -914-1001Pat#15019519 Rev. A December 2010,Illumina。
  5. Ku,J.L.,Jeon,Y.K.,Park,J.G。(2011)。 甲基化特异性PCR 。 Methods Mol Biol 791:23-32。
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引用:Zeller, C., Masrour, N., Patel, N., Dai, W., Wilhelm-Benartzi, C. and Brown, R. (2013). DNA Methylation Profiling Using Infinium Methylation Assay. Bio-protocol 3(10): e777. DOI: 10.21769/BioProtoc.777.