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

Denne undersøgelse blev støttet af Det Svenske Forskningsråd, Kræftens Bekæmpelse, Knut- og Alice Wallenbergfonden (KAW), og VD VästraGötalandsregionen.

Den etiske godkendelse til indsamling af CLL-prøverne er fra 2007-05-21, med følgende registreringsnummer: EPN Gbg dnr 239/07. Alle CLL-patienter blev diagnosticeret ifølge nyligt reviderede kriterier 8 , og prøverne blev samlet på diagnosetidspunktet. Patienterne i undersøgelsen blev inkluderet fra forskellige hæmatologiske afdelinger i den vestlige del af Sverige efter skriftligt samtykke. Kun prøver af CLL perifert blodmononukleær celle (PBMC) med en tumorprocent af leukæmiske celler...

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MBD-seq er en omkostningseffektiv immunpræcipitationsbaseret teknik, der kan bruges til at studere methyleringsmønstre med komplet gennembrydende dækning. Både MeDIP-seq (methyleret DNA-immunpræcipitation efterfulgt af sekventering) og MBD-seq resulterer i berigelse af CpG-rich methyleret DNA. Imidlertid viser MBD-seq mere affinitet mod binding til stærkt CpG-rige regioner, når de sammenlignes med MeDIP-seq 19 . Ved anvendelse af et methylbindende berigelsessæt kan man fraktionere DNA&#39...

Anvendelsen af ​​næste generations sekventeringsteknikker til analyse af globale DNA-methyleringsprofiler er blevet stadig mere populært i de seneste år. Genom-brede methyleringsassays, herunder mikroarray- og ikke-mikroarray-baserede metoder, blev udviklet baseret på følgende: bisulfitomdannelsen af ​​genomisk DNA, methyleringskänslig restriktionsenzymfordøjelse og immunpræcipitation af methyleret DNA under anvendelse af methyl CpG-specifikke antistoffer . Aberrant DNA-methylering er et kendetegn ved leukæmi og lymfomer, herunder kronisk lymfocytisk leukæmi (CLL). Tidligere har flere grupper, herunder vores, karakteriseret DNA-methyleringsprofilerne fra forskellige CLL-prognostiske undergrupper og normale, sunde B-cellekontroller under anvendelse af bisulfitomdannelsen af ​​genomisk DNA efterfulgt af mikromarraybaserede metoder eller helgenomsekvensering 1 , 2 , 3 , &lt;Sup class = &quot;xref&quot;&gt; 4. Bisulfitomdannelsen af ​​genomisk DNA fører til deaminering af umodificerede cytosiner til uracil, hvilket efterlader de modificerede methylerede cytosiner i genomet. Efter omdannelse kan methyleringsstatusen for DNA&#39;et bestemmes ved PCR-amplifikation og sekventering under anvendelse af forskellige kvantitative eller kvalitative metoder, såsom mikro-array-baseret eller helgenoms-bisulfit-sekventering (WGBS). Selvom bisulfit-konverteringsbaserede metoder har mange fordele og er meget udbredt i forskellige kræfttyper til analyse af DNA-methyleringsniveauer, er der nogle ulemper forbundet med denne teknik. WGBS sekventering tillader single-base-pair opløsning med lavere mængder af DNA og er den bedst egnede mulighed for at analysere et stort antal prøver. Denne metode undlader imidlertid at differentiere modifikationerne mellem 5 mC og 5 hmC niveauerne i genomet 5 , 6 . Desuden tilbyder microarray-baserede metoder ikke komplet cOverage af genomet. I en nylig undersøgelse fra vores laboratorium 7 blev immunpræcipitationsbaserede metoder, snarere end bisulfitkonvertering, anvendt til at identificere højt CpG-rige, differentielt methylerede regioner i global skala hos CLL-patienter og normale sunde kontroller. Inmethyl-CpG-bindingsdomæne (MBD) næste generations sekventering (MBD-seq) afhænger berigelsen af ​​dobbeltstrenget fragmenteret DNA af graden af ​​CpG-methylering. Denne fremgangsmåde kan overvinde ulemperne ved bisulfitomdannelsesmetoden og kan også tilvejebringe genomomfattende dækning af CpG-methylering på en upartisk og PCR-uafhængig måde. Derudover kan MBD-seq i modsætning til bisulfit-konverteringsbaserede mikroarray-metoder anvendes til at analysere gentagelseselementers methyleringsstatus, såsom lange interspersed-nukleare elementer (LINE&#39;er), korte interspersed-nukleare elementer (SINEs), lange terminale gentagelser (LTRS), Osv . Imidlertid sammenlignet med bisulfitomdannelsesmetoder,En MBD-seq-protokol kræver en relativt stor mængde input-DNA. Kvaliteten af ​​sekventeringen læser også, og dataene afhænger af specificiteten, affiniteten og kvaliteten af ​​de anvendte antistoffer. Den nuværende undersøgelse forklarer en detaljeret MBD-seq-protokol til berigelse af methyleret DNA til næste generations sekventering. Den anvender et kommercielt methyleret DNA-bindings berigelse kit (opført i Materialetabellen ) samt en beregningsrørledning til visualisering og fortolkning af methylerings-sekventeringsdata for at identificere CLL-specifikke hyper- og hypomethylerede regioner sammenlignet med normale sunde kontroller. I grund og grund gør denne metode brug af MBD&#39;ens evne til human MBD2-proteininteraktion med methylerede CpG&#39;er til ekstraktion af DNA beriget med methylerede CpG&#39;er, og dette efterfølges af høj-gennemstrømningssekvenseringen af ​​methyleret DNA.

<table border="0" cellpadding="0" cellspacing="0" width="626">
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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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			<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>
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			<td height="51" style="height:51px;width:216px;">TE buffer PH 8</td>
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			<td style="width:119px;">93283</td>
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			<td height="42" style="height:42px;width:216px;">Bioruptor standard sonication device</td>
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			<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>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3 M Sodium acetate</td>
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			<td>C03030002</td>
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			<td height="42" style="height:42px;width:216px;">E-gel iBase safe imager combo kit</td>
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			<td style="width:119px;">G6465EU</td>
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			<td height="42" style="height:42px;width:216px;">E-gel 2% Agarose gels</td>
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			<td style="width:119px;">G441002</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>
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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>
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			<td height="21" style="height:21px;width:216px;">Vortex mixer</td>
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			<td style="width:119px;">12620-848</td>
			<td></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>
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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>
		</tr>
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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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		</tr>
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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>
		</tr>
		<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>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Illumina Hiseq2000 Platform</td>
			<td style="width:124px;">Illumina</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Water&#160; Bath</td>
			<td style="width:124px;">Grant</td>
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			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Heat block</td>
			<td style="width:124px;">grant</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Tube rotater</td>
			<td style="width:124px;">Labquake</td>
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</table>

comprehensive dna methylation analysis using a methyl-cpg-binding domain capture-based method in chronic lymphocytic leukemia patients

Rollen af ​​lange ikke-kodende RNA&#39;er (lncRNA&#39;er) i kræft kommer frem i forgrunden på grund af den voksende interesse for at forstå deres mekaniske funktioner under udvikling af kræft og progression. På trods af dette er den globale epigenetiske regulering af lncRNA&#39;er og gentagne sekvenser i kræft ikke blevet undersøgt godt, især i kronisk lymfocytisk leukæmi (CLL). Denne undersøgelse fokuserer på en unik tilgang: Den immunpræcipitationsbaserede indfangning af dobbeltstrengede, methylerede DNA-fragmenter ved anvendelse af methyl-bindende domæne (MBD) proteiner efterfulgt af næste generations sekventering (MBD-seq). CLL patientprøver tilhørende to prognostiske undergrupper (5 IGVH-muterede prøver + 5 IGVH-ikke-muterede prøver) blev anvendt i dette studie. Analyse afslørede 5.800 hypermethylerede og 12.570 hypomethylerede CLL-specifikke differentielt methylerede gener (cllDMG&#39;er) sammenlignet med normale sunde kontroller. Det er vigtigt, at disse resultater identificerede adskillige CLL-specifikke, differentielt methylerede lncRNA&#39;er, rePetitive elementer og proteinkodende gener med potentiel prognostisk værdi. Dette arbejde beskriver en detaljeret protokol for en MBD-seq og bioinformatik pipeline udviklet til en omfattende analyse af globale methyleringsprofiler i højt CpG-rige regioner ved brug af CLL patientprøver. Endelig valideredes et proteinkodende gen og et lncRNA under anvendelse af pyrosequencing, hvilket er en meget kvantitativ metode til analyse af CpG-methyleringsniveauer for yderligere at bekræfte resultaterne fra MBD-seq-protokollen.

MBD-seq blev for nylig udført på CLL-patienter og matchede, normale, sunde kontroller til identifikation af CLL-specifikke differentielt hyper- og hypomethylerede gener 7 . Den eksperimentelle og bioinformatiske rørledning, der anvendes til analyse af data genereret fra CLL og normale sunde prøver, er vist i figur 1A og 1B . Disse analyser identificerede adskillige CLL-specifikke differentielt methylerede regioner...

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

Dette arbejde beskriver en optimeret methyl-CpG-bindingsdomæne (MBD) sekventeringsprotokol og en beregningsrørledning til identifikation af differentielt methylerede CpG-rige regioner hos patienter med kronisk lymfocytisk leukæmi (CLL).

Omfattende DNA-methyleringsanalyse ved anvendelse af en methyl-CpG-bindingsdomæne-capture-baseret metode i patienter med kronisk lymfocytisk leukæmi

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

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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

Bestemmelse af Optimal kromosomale placering for en DNA Element i Escherichia coli ved hjælp af en roman Transposon-medieret tilgang

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

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

Immunfarvning for DNA ændringer: beregningsmæssige analyse af Konfokal billeder

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

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

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

Profilering DNA replikation Timing ved hjælp af zebrafisk som en In Vivo modelsystem.

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

Kandidat gen test i kliniske kohorteundersøgelser med multipleksede genotypebestemmelse og massespektrometri

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

Metoder til nøjagtig detektering af mitokondrie-DNA methylering

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

Kvantitativ analyse af Alternative Pre-mRNA-Splicing i mus hjernen sektioner ved hjælp af RNA In Situ hybridisering Assay

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 FÃƒÆ’Ã‚Â¶rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Single-molekyle Förster Resonance energioverførsel metoder til real-time undersøgelse af Holliday Junction resolution af GEN1

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

Undersøgelse af den transkriptionelle rolle af en RUNX1 intronic LYDDÆMPER af crispr/Cas9 ribonucleoprotein i akutte myeloid leukæmiceller

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-2ÃƒÂ¢Ã¢â€šÂ¬Ã‚Â²-Deoxyuridine IdU with Cell Cycle Markers to Establish Cell Cycle Phases in a Mass Cytometry Platform

Anvendelse af pyrimidinanalogen, 5-Iodo-2′-deoxyuridin (IdU) med cellecyklusmarkører til at etablere cellecyklusfaser i en massecytometriplatform

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

LINE-1 MethyleringAnalyse i mesenkymale stamceller behandlet med osteosarkom-afledte ekstracellulære vesikler

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