Name: comprehensive dna methylation analysis using a methyl-cpg-binding domain capture-based method in chronic lymphocytic leukemia patients
Uploaded: 2017-06-16T13:00:00
Description: Watch this Scientific Journal Video about Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients at JoVE.com

瑞典研究委员会，瑞典癌症协会，Knut和爱丽丝·瓦伦贝格基金会（KAW）以及FoUVästraGötalandsregionen都支持这项研究。

收集CLL样本的道德准许是2007-05-21，具有以下注册号：EPN Gbg dnr 239/07。所有CLL患者根据最近修订的标准8诊断，并在诊断时收集样品。在获得书面同意后，研究中的患者被纳入瑞典西部不同血液学部门。本研究仅选择了白血病细胞百分率≥70％的CLL外周血单个核细胞（PBMC）样本。 准备工作<ol><li>根据制造商的说明书，使用商业分离试剂盒（参见材料表</s...

<ol>
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M., Lohse, M., Usadel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trimmomatic:+a+flexible+trimmer+for+Illumina+sequence+data.">Trimmomatic: a flexible trimmer for Illumina sequence data.</a> Bioinformatics. 30 (15), 2114-2120 (2014).</li><li>Langmead, B., Trapnell, C., Pop, M., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+and+memory-efficient+alignment+of+short+DNA+sequences+to+the+human+genome.">Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.</a> Genome Biol. 10 (3), R25 (2009).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model-based+analysis+of+ChIP-Seq+(MACS).">Model-based analysis of ChIP-Seq (MACS).</a> Genome Biol. 9 (9), R137 (2008).</li><li>Quinlan, A. R., Hall, I. 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MBD-seq是一种经济有效的基于免疫沉淀的技术，可用于研究具有完整全基因组范围覆盖的甲基化模式。 MeDIP seq（甲基化DNA免疫沉淀随后测序）和MBD-seq均导致富含CpG的甲基化DNA的富集。然而，与MeDIP seq 19相比，MBD-seq对结合高度富含CpG的区域具有更强的亲和力。使用甲基结合富集试剂盒，可以分别通过高盐和低盐缓冲液洗脱DNA将DNA分馏成高CpG和低CpG富集区。在该研究中，仅进行单?...

近年来，使用下一代测序技术分析全球DNA甲基化谱已越来越受欢迎。基于全基因组的甲基化测定，包括基于微阵列和非微阵列的方法，基于以下方法开发：基因组DNA的亚硫酸氢盐转化，甲基化敏感的限制酶消化和甲基化DNA的免疫沉淀，使用甲基CpG特异性抗体。 异常DNA甲基化是白血病和淋巴瘤的标志之一，包括慢性淋巴细胞性白血病（CLL）。早些时候，包括我们在内的几个组织表征不同CLL预后亚组和正常健康B细胞对照的DNA甲基化谱，使用基因组DNA的亚硫酸氢盐转化，然后是基于微阵列的方法或全基因组测序1,2,3 ， &lt;sup class =“xref”&gt; 4。基因组DNA的亚硫酸氢盐转化导致未修饰的胞嘧啶脱氨酸尿嘧啶，留下基因组中修饰的甲基化胞嘧啶。一旦转化，DNA的甲基化状态可以通过PCR扩增和使用不同的定量或定性方法如基于微阵列或全基因组亚硫酸氢盐测序（WGBS）的测序来测定。尽管基于亚硫酸氢盐转化的方法具有许多优点，并且广泛用于不同的癌症类型以分析DNA甲基化水平，但与该技术相关的一些缺点是。 WGBS测序允许单碱基对分辨率较低的DNA，是分析大量样品的最佳选择。然而，这种方法不能区分基因组中5mC和5mmC水平之间的修饰。此外，基于微阵列的方法不提供完整的c基因组超量。 在我们实验室7最近的一项研究中，基于免疫沉淀的方法，而不是亚硫酸氢盐转化，用于在CLL患者和正常健康对照组中在全球范围内鉴定高度富含CpG的差异甲基化区域。在甲基CpG结合域（MBD）下一代测序（MBD-seq）中，双链片段化DNA的富集取决于CpG甲基化程度。该方法可以克服亚硫酸氢盐转化方法的缺点，并且还可以以无偏差和不依赖PCR的方式提供CpG甲基化的全基因组覆盖。此外，与基于亚硫酸氢盐转化的微阵列方法不同，MBD-seq可用于分析重复元件的甲基化状态，例如长散布核元素（LINE），短散点核元素（SINE），长末端重复序列（LTRS） 等等然而，与亚硫酸氢盐转化方法相比，MBD-seq协议需要相当大量的输入DNA。此外，测序的质量读数和数据取决于所用抗体的特异性，亲和性和质量。 目前的研究解释了详细的MBD-seq方案来丰富用于下一代测序的甲基化DNA。它使用商业甲基化DNA结合浓缩试剂盒（在材料表中列出 ），以及计算流程，用于显示和解释甲基化测序数据，以鉴定与正常健康对照相比的CLL特异性高和低甲基化区域。基本上，这种方法利用人类MBD2蛋白与甲基化CpG相互作用的MBD的能力来提取富含甲基化CpG的DNA，其次是甲基化DNA的高通量测序。

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			<td height="21" style="height:21px;width:216px;">Dneasy Blood and tissue kit</td>
			<td style="width:124px;">Qaigen</td>
			<td style="width:119px;">69504</td>
			<td style="width:167px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lymphoprep solution</td>
			<td style="width:124px;">A X I S-S H I E L D</td>
			<td style="width:119px;">1114544</td>
			<td style="width:167px;"></td>
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			<td height="42" style="height:42px;width:216px;">Nano drop 2000</td>
			<td style="width:124px;">Thermo Fischersceintific</td>
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			<td height="51" style="height:51px;width:216px;">TE buffer PH 8</td>
			<td style="width:124px;">Sigma aldrich</td>
			<td style="width:119px;">93283</td>
			<td style="width:167px;"></td>
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			<td height="42" style="height:42px;width:216px;">Bioruptor standard sonication device</td>
			<td style="width:124px;">Diagenode</td>
			<td style="width:119px;">UCD-200</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TPX bioruptor tubes 1.5ml</td>
			<td style="width:124px;">Diagenode</td>
			<td style="width:119px;">C30010010-300</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3 M Sodium acetate</td>
			<td style="width:124px;">Diagenode</td>
			<td>C03030002</td>
			<td style="width:167px;"></td>
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			<td height="42" style="height:42px;width:216px;">E-gel iBase safe imager combo kit</td>
			<td style="width:124px;">Thermo Fischersceintific</td>
			<td style="width:119px;">G6465EU</td>
			<td style="width:167px;"></td>
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			<td height="42" style="height:42px;width:216px;">E-gel 2% Agarose gels</td>
			<td style="width:124px;">Thermo Fischersceintific</td>
			<td style="width:119px;">G441002</td>
			<td style="width:167px;"></td>
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			<td height="42" style="height:42px;width:216px;">Methylminer Methylated DNA enrichment kit</td>
			<td style="width:124px;">Thermo Fischersceintific</td>
			<td style="width:119px;">ME10025</td>
			<td style="width:167px;"></td>
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			<td height="42" style="height:42px;width:216px;">Labquake Tube Shaker/Rotators</td>
			<td style="width:124px;">Thermo Fischersceintific</td>
			<td style="width:119px;">415110</td>
			<td style="width:167px;"></td>
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			<td height="42" style="height:42px;width:216px;">Dynal MPC-S</td>
			<td style="width:124px;">Thermo Fischersceintific</td>
			<td style="width:119px;">A13346</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:216px;">Vortex mixer</td>
			<td style="width:124px;">VWR</td>
			<td style="width:119px;">12620-848</td>
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			<td height="21" style="height:21px;width:216px;">Absolute Ethanol</td>
			<td style="width:124px;">Any company</td>
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			<td height="21" style="height:21px;width:216px;">70% Ethanool</td>
			<td style="width:124px;">Any company</td>
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			<td height="21" style="height:21px;width:216px;">DNAse free water</td>
			<td style="width:124px;">Milli Q</td>
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			<td style="width:167px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DNA precipitant (3M sodium acetate)</td>
			<td style="width:124px;">Diagenode</td>
			<td style="width:119px;">C03030002</td>
			<td style="width:167px;"></td>
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			<td height="42" style="height:42px;width:216px;">Safe seal 1.5ml eppendorf tubes</td>
			<td style="width:124px;">Eppendorf</td>
			<td style="width:119px;">4036-3204</td>
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			<td height="42" style="height:42px;width:216px;">Qubit dsDNA HS Assay Kit</td>
			<td style="width:124px;">Thermo Fischersceintific</td>
			<td style="width:119px;">Q32851</td>
			<td style="width:167px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Qubit 0.5ml tubes</td>
			<td style="width:124px;">Thermo Fischersceintific</td>
			<td style="width:119px;">Q32856</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Qubit</td>
			<td style="width:124px;">Thermo Fischersceintific</td>
			<td style="width:119px;">Q32866</td>
			<td></td>
		</tr>
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			<td height="20" style="height:20px;width:216px;">Illumina Hiseq2000 Platform</td>
			<td style="width:124px;">Illumina</td>
			<td style="width:119px;"></td>
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			<td height="20" style="height:20px;width:216px;">Water&#160; Bath</td>
			<td style="width:124px;">Grant</td>
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			<td height="20" style="height:20px;width:216px;">Heat block</td>
			<td style="width:124px;">grant</td>
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			<td height="20" style="height:20px;width:216px;">Tube rotater</td>
			<td style="width:124px;">Labquake</td>
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comprehensive dna methylation analysis using a methyl-cpg-binding domain capture-based method in chronic lymphocytic leukemia patients

长期非编码RNA（lncRNA）在癌症中的作用已经走在前列，因为在癌症发展和进展期间越来越需要了解其机械功能。尽管如此，在癌症中lncRNA和重复序列的全球表观遗传调控尚未得到很好的研究，特别是在慢性淋巴细胞性白血病（CLL）中。本研究着重于一种独特的方法：使用甲基结合结构域（MBD）蛋白进行基于免疫沉淀的双链甲基化DNA片段捕获，随后进行下一代测序（MBD-seq）。本研究使用属于两个预后亚组（5个IGVH突变样本+ 5个IGVH未突变样本）的CLL患者样本。与正常健康对照相比，分析显示5,800个高甲基化和12,570个低甲基化的CLL特异性差异甲基化基因（cllDMG）。重要的是，这些结果确定了几种CLL特异性差异甲基化的lncRNA具有潜在预后价值的蛋白质编码基因。该工作概述了MBD-seq和生物信息学管道的详细协议，用于使用CLL患者样本全面分析高CpG丰富地区的全球甲基化谱。最后，使用焦磷酸测序验证蛋白质编码基因和lncRNA，这是一种高度定量的方法来分析CpG甲基化水平，以进一步证实MBD-seq方案的发现。

最近对CLL患者和匹配的正常健康对照进行了MBD-seq，以鉴定CLL特异性差异超低和低甲基化基因7 。用于分析CLL和正常健康样品产生的数据的实验和生物信息管道如图1A和1B所示 。这些分析确定了几个CLL特异性差异甲基化区域（cllDMR），与对照样品相比，IGHV突变和IGHV未突变样品显着高/低甲基化，p值&lt;0.00001。 <strong class="xf...

Watch this Scientific Journal Video about Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients at JoVE.com

Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients

该工作描述了优化的甲基CpG结合结构域（MBD）测序方案和用于鉴定慢性淋巴细胞性白血病（CLL）患者中差异甲基化CpG富集区的计算管道。

在慢性淋巴细胞性白血病患者中使用甲基CpG结合域捕获法进行综合DNA甲基化分析

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during ...

The overall goal of this procedure is to study global methylation profiling and identify differentially methylated CpG-rich regions in chronic lymphocytic leukemia patients using next-generation sequencing. This method can help answer key questions in the epigenetics field such as identification of the potential CLL-specific signature genes disease pathogenesis, and prognosis. The main advantage of this technique is that it provides complete genome-wide coverage of CpG methylation in an unbiased and PCR-independent manner.To begin, use a commercially available DNA extraction kit to isolate genomic DNA from the patient and normal peripheral blood mononuclear cell samples according to the manufacturer's protocol. After quantifying the genomic DNA as described in the text protocol, dilute 5 micrograms of genomic DNA from each sample, to a total of 200 microliters using TE buffer, giving a final concentration of 25 nanograms per microliter. Next, perform sonication in specially-designed tubes, using a sonicator, for a total of 30 cycles of 30 seconds on and 30 seconds off.After every five cycles, briefly spin the tubes to collect the samples to the bottom. Check the sonication range for all samples using DNA electrophoresis as described in the text protocol. To prepare the magnetic streptavidin beads, first re-suspend them from the stock tube provided by the kit.Gently pipette the beads up and down to obtain a homogenous suspension. For each five micrograms of fragmented DNA sample, place 50 microliters of the beads into separate, clean and labeled 1.5-milliliter tubes. Add 50 microliters of 1X bead wash buffer to reach a final volume of 100 microliters.Place the tubes on a magnetic stand for one minute to allow all the magnetic beads to concentrate on the inner wall of the tube facing the magnet. Remove the liquid without touching the beads, using a 200 microliter pipette. Then, remove the tubes from the magnetic stand, add 250 microliters of 1X bead wash buffer, and mix the beads gently with a pipette.After repeating these steps at least four times for all samples, finally resuspend the samples in 250 microliters of 1X bead wash buffer. Keep the samples on ice. To bind the MBD-biotin protein to the washed beads, first add 35 microliters of protein to separate tubes and bring the total volume up to 250 microliters using 1X bead wash buffer.Add 250 microliters of diluted MBD protein to the 250 microliters of washed beads and leave them on end-to-end rotation at room temperature. After mixing the beads in protein for one hour, wash the protein-bound magnetic streptavidin beads by placing the tubes on the magnetic stand for one minute. Remove the liquid without touching the beads using a pipette.Then, add 250 microliters of 1X bead wash buffer and place the tubes on a rotation mixer for five minutes at room temperature. Repeat the washing step two more times before re-suspending the washed MBD-biotin beads in 200 microliters of 1X bead wash buffer, making the beads ready for methylated DNA capture. In a clean 1.5-milliliter DNase-free tube, add 100 microliters of milliliter bead wash buffer and 180 microliters of the fragmented genomic DNA.Bring the final volume to 500 microliters using DNase-free water. Add 380 microliters of DNase-free water to the remaining 20 liters of the fragmented genomic DNA. Freeze the samples to later process them further and use them as input DNA controls.Place the tubes containing washed MBD-biotin beads on a magnetic stand for one minute, and remove the liquid without disturbing the beads. Then, add 500 microliters of fragmented genomic DNA diluted in bead wash buffer. Seal all the tubes tightly with paraffin film and leave them overnight at 4 degrees Celsius on an end-to-end rotation stand at 8 to 10 rotations per minute.After the DNA and MBD bead binding reaction, place the tubes on the magnetic rack for one minute to concentrate all the beads on the inner wall of the tube. Remove the supernatant liquid with a pipette without touching the beads, and save this unbound DNA sample fraction on ice. Then add 200 microliters of 1X bead wash buffer to the beads and place the tubes on the rotating stand for three minutes at room temperature.After removing the liquid using the magnetic stand, repeat the wash another two times to remove the residual unbound DNA. After the final wash, add 200 microliters of high-salt elution buffer provided in the kit to elute the DNA. Then, place the tubes on the rotating stand for 15 minutes at room temperature.Following incubation, place them on the magnetic stand for one minute and use a pipette to carefully transfer the supernatant to a new, clean 1.5-milliliter tube. Next, add 200 microliters of high-salt elution buffer to the beads, and repeat the elution by rotating the tubes at room temperature for an additional 15 minutes. Add the second elute to the same tube containing the 200 microliters of the first elute.To precipitate the DNA, add one microliter of glycogen, 40 microliters of three molar sodium acetate, pH 5.2, and 800 microliters of ice-cold absolute ethanol to 400 microliters of eluted DNA. Also add the reagents to the previously frozen 400 microliters of input DNA control samples. Mix the tubes well by vortexing before incubating them at minus 80 degrees Celsius overnight.The next day, centrifuge the tubes at 12, 000 times g and 4 degrees Celsius for 30 minutes. Discard the supernatant carefully without disturbing the pellet. Then, add 500 microliters of 70%ethanol and vortex the tubes.After centrifuging the tubes again using the same conditions but for 15 minutes, remove the supernatant by carefully using a pipette. Then, centrifuge the tubes at maximum speed for one minute at room temperature, and remove the residual ethanol completely using a pipette tip. Air-dry the pellet for five minutes at room temperature.Finally, add 10 microliters of DNase-free water to the DNA pellet before proceeding to the methylated DNA quantification, MBD sequencing, and analysis as described in the text protocol. The analysis identified several differentially hyper-and hypomethylated regions enriched in CLL samples compared to normal controls. These differentially methylated regions were mapped to different classes of protein coding and noncoding genes.Finally, the data analysis showed significant overlap between the CLL-associated differentially methylated regions obtained from two different normal control comparisons. Here, all the CpG sites located in target regions of two differentially methylated genes were validated in a large number of CLL samples using pyrosequencing. The optimum range of sonication required for this method is illustrated here.This range of fragmented DNA is ideal and more suitable for next-generation sequencing purposes. Once mastered, this technique can be done in two days if it is done properly. While attempting this procedure, the crucial factors that affect the final DNA recovery are the quality and the amount of the input DNA used and the range of the fragmented DNA after sonication.We decided to use this method because it is the first CLL global methylation study that is based on the immunoprecipitation for enriching methylated DNA covering all the methylated regions across the genome. The implications of this technique extend towards prognosis or therapy because the identified differentially methylated signature genes could serve as global normal biomarkers and epigenetic targets for therapy. Following this procedure, the enriched methylated DNA can be used for other downstream applications like hybridizing or doing microarrays to easily compare the metalomes between two samples or disease entities using a fixed set of CpG sites present on the arrays.Finally, this is a cost-effective technique to analyze large number of patient samples for methylated sequences across the genome, including annotated sequences spanning protein-coding genes as well as non-annotated sequences spanning repetitive elements and long non-coding RNAs.

Research

JoVE Journal

Genetics

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

用新的转介导法测定大肠杆菌中 DNA 元素的最佳染色体位置 (s)

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

科研

遗传学

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

应用多重基因分型和质谱法进行临床队列研究的候选基因检测

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

线粒体 DNA 甲基化的精确检测方法

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

RNA原位杂交法定量分析小鼠脑切片中的替代前 mRNA 拼接方法

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Single-Molecule F&ouml;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Single-Molecule F&#246;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

单分子Fürster共振能量传递方法，用于GEN1对霍利迪结分辨率进行实时调查

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

CRISPR/Cas9核糖核酸蛋白在急性骨髓性白血病细胞中的RUNX1电子消光器的转录作用研究

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...

Use of the Pyrimidine Analog, 5-Iodo-2&prime;-Deoxyuridine (IdU) with Cell Cycle Markers to Establish Cell Cycle Phases in a Mass Cytometry Platform

Use of the Pyrimidine Analog, 5-Iodo-2&#8242;-Deoxyuridine IdU with Cell Cycle Markers to Establish Cell Cycle Phases in a Mass Cytometry Platform

使用嘧啶类似物5-碘-2′-脱氧尿苷（IdU）和细胞周期标志物在质谱细胞术平台中建立细胞周期期

The regulation of cell cycle phase is an important aspect of cellular proliferation and homeostasis. Disruption of the regulatory mechanisms governing the ...

LINE-1 Methylation Analysis in Mesenchymal Stem Cells Treated with Osteosarcoma-Derived Extracellular Vesicles

用骨肉瘤衍生细胞外囊泡处理的美相干细胞中的LINE-1甲基化分析

Methylation-specific probe amplification (MSPA) is a simple and robust technique that can be used to detect relative differences in methylation levels of ...

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

使用麦格纳波特奥里扎伊的色度免疫沉淀测序方法对希斯通修饰分布进行全基因组分析

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription ...