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

Questo studio è stato sostenuto dal Consiglio di ricerca svedese, dalla Società cancro svedese, dalla fondazione Knut e Alice Wallenberg (KAW) e da FoU VästraGötalandsregionen.

L&#39;approvazione etica per la raccolta dei campioni CLL è dal 2007-05-21, con il seguente numero di registrazione: EPN Gbg dnr 239/07. Tutti i pazienti affetti da CLL sono stati diagnosticati in base ai criteri di revisione recenti 8 , ei campioni sono stati raccolti al momento della diagnosi. I pazienti dello studio sono stati inclusi in diversi reparti di ematologia nella parte occidentale della Svezia dopo che era stato ottenuto il consenso scritto. In questo studio sono stati selezionati s...

<ol>
	<li>Kanduri, M., 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=Differential+genome-wide+array-based+methylation+profiles+in+prognostic+subsets+of+chronic+lymphocytic+leukemia.">Differential genome-wide array-based methylation profiles in prognostic subsets of chronic lymphocytic leukemia.</a> Blood. 115 (2), 296-305 (2010).</li><li>Cahill, N., 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=450K-array+analysis+of+chronic+lymphocytic+leukemia+cells+reveals+global+DNA+methylation+to+be+relatively+stable+over+time+and+similar+in+resting+and+proliferative+compartments.">450K-array analysis of chronic lymphocytic leukemia cells reveals global DNA methylation to be relatively stable over time and similar in resting and proliferative compartments.</a> Leukemia. 27 (1), 150-158 (2013).</li><li>Kanduri, M., 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=Distinct+transcriptional+control+in+major+immunogenetic+subsets+of+chronic+lymphocytic+leukemia+exhibiting+subset-biased+global+DNA+methylation+profiles.">Distinct transcriptional control in major immunogenetic subsets of chronic lymphocytic leukemia exhibiting subset-biased global DNA methylation profiles.</a> Epigenetics. 7 (12), 1435-1442 (2012).</li><li>Kulis, M., 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=Epigenomic+analysis+detects+widespread+gene-body+DNA+hypomethylation+in+chronic+lymphocytic+leukemia.">Epigenomic analysis detects widespread gene-body DNA hypomethylation in chronic lymphocytic leukemia.</a> Nat Genet. 44 (11), 1236-1242 (2012).</li><li>Booth, M. J., 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=Quantitative+sequencing+of+5-methylcytosine+and+5-hydroxymethylcytosine+at+single-base+resolution.">Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution.</a> Science. 336 (6083), 934-937 (2012).</li><li>Yu, M., 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=Base-resolution+analysis+of+5-hydroxymethylcytosine+in+the+mammalian+genome.">Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome.</a> Cell. 149 (6), 1368-1380 (2012).</li><li>Subhash, S., Andersson, P. O., Kosalai, S. T., Kanduri, C., Kanduri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+DNA+methylation+profiling+reveals+new+insights+into+epigenetically+deregulated+protein+coding+and+long+noncoding+RNAs+in+CLL.">Global DNA methylation profiling reveals new insights into epigenetically deregulated protein coding and long noncoding RNAs in CLL.</a> Clin Epigenetics. 8, 106 (2016).</li><li>Hallek, M., 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=Guidelines+for+the+diagnosis+and+treatment+of+chronic+lymphocytic+leukemia:+a+report+from+the+International+Workshop+on+Chronic+Lymphocytic+Leukemia+updating+the+National+Cancer+Institute-Working+Group+1996+guidelines.">Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines.</a> Blood. 111 (12), 5446-5456 (2008).</li><li>De Meyer, T., 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=Quality+evaluation+of+methyl+binding+domain+based+kits+for+enrichment+DNA-methylation+sequencing.">Quality evaluation of methyl binding domain based kits for enrichment DNA-methylation sequencing.</a> PLoS One. 8 (3), e59068 (2013).</li><li>Bolger, A. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BEDTools:+a+flexible+suite+of+utilities+for+comparing+genomic+features.">BEDTools: a flexible suite of utilities for comparing genomic features.</a> Bioinformatics. 26 (6), 841-842 (2010).</li><li>Subhash, S., Kanduri, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GeneSCF:+a+real-time+based+functional+enrichment+tool+with+support+for+multiple+organisms.">GeneSCF: a real-time based functional enrichment tool with support for multiple organisms.</a> BMC Bioinformatics. 17 (1), 365 (2016).</li><li>Liao, Y., Smyth, G. K., Shi, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=featureCounts:+an+efficient+general+purpose+program+for+assigning+sequence+reads+to+genomic+features.">featureCounts: an efficient general purpose program for assigning sequence reads to genomic features.</a> Bioinformatics. 30 (7), 923-930 (2014).</li><li>Robinson, M. D., McCarthy, D. J., Smyth, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=edgeR:+a+Bioconductor+package+for+differential+expression+analysis+of+digital+gene+expression+data.">edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.</a> Bioinformatics. 26 (1), 139-140 (2010).</li><li>Martinelli, S., 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=ANGPT2+promoter+methylation+is+strongly+associated+with+gene+expression+and+prognosis+in+chronic+lymphocytic+leukemia.">ANGPT2 promoter methylation is strongly associated with gene expression and prognosis in chronic lymphocytic leukemia.</a> Epigenetics. 8 (7), 720-729 (2013).</li><li>Kopparapu, P. K., 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=Epigenetic+silencing+of+miR-26A1+in+chronic+lymphocytic+leukemia+and+mantle+cell+lymphoma:+Impact+on+EZH2+expression.">Epigenetic silencing of miR-26A1 in chronic lymphocytic leukemia and mantle cell lymphoma: Impact on EZH2 expression.</a> Epigenetics. 11 (5), 335-343 (2016).</li><li>Robinson, M. D., 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=Evaluation+of+affinity-based+genome-wide+DNA+methylation+data:+effects+of+CpG+density,+amplification+bias,+and+copy+number+variation.">Evaluation of affinity-based genome-wide DNA methylation data: effects of CpG density, amplification bias, and copy number variation.</a> Genome Res. 20 (12), 1719-1729 (2010).</li></ol>

MBD-seq è una tecnica basata su costo-efficacia basata sull&#39;immunoprecipitazione che può essere utilizzata per studiare i modelli di metilazione con copertura completa a livello genomico. Sia MeDIP seq (immunoprecipitazione metilata DNA seguita da sequenziamento) e MBD-seq provocano l&#39;arricchimento di DNA metilato ricco di CpG. Tuttavia, MBD-seq mostra maggiore affinità verso l&#39;adesione a regioni ricche di CpG molto elevate rispetto a MeDIP seq 19 . Utilizzando un kit di arricchime...

L&#39;utilizzo di tecniche di sequenziamento di nuova generazione per l&#39;analisi dei profili di metilazione del DNA globale è diventato sempre più popolare negli ultimi anni. I metodi di metilazione a livello genomico, compresi metodi basati su microarray e non microarray, sono stati sviluppati in base a quanto segue: la conversione di bisulfite di DNA genomico, digestioni enzimatiche di restrizione sensibile alla metilazione e l&#39;immunoprecipitazione del DNA metilato usando anticorpi specifici di metil CpG . La metilazione aberrante del DNA è uno dei segni distintivi della leucemia e dei linfomi, inclusa la leucemia linfocitica chirurgica (CLL). In precedenza, diversi gruppi compresi i nostri caratterizzavano i profili di metilazione del DNA di diversi sottogruppi CLL e dei normali e sani controlli delle cellule B usando la conversione bisulfa del DNA genomico seguita da metodi basati su micro-array o sequenziamenti interi genomi 1 , 2 , 3 , &lt;Sup class = &quot;xref&quot;&gt; 4. La conversione di bisulfite del DNA genomico conduce alla deaminazione di citosine non modificate all&#39;uracil, lasciando le citosine metilate modificate nel genoma. Una volta convertito, lo stato di metilazione del DNA può essere determinato mediante amplificazione e sequenza PCR utilizzando diversi metodi quantitativi o qualitativi, come il sequenziamento di bisulfite a base di micro-array o intero genoma (WGBS). Sebbene i metodi basati sulla conversione di bisolfito abbiano molti vantaggi e sono ampiamente usati in diversi tipi di cancro per analizzare i livelli di metilazione del DNA, ci sono alcuni inconvenienti associati a questa tecnica. Il sequenziamento di WGBS consente la risoluzione di una coppia di basi con quantità minori di DNA ed è l&#39;opzione migliore per analizzare un gran numero di campioni. Tuttavia, questo metodo non riesce a differenziare le modifiche tra i livelli di 5 mC e 5 hmC nel genoma 5 , 6 . Inoltre, i metodi basati su microarray non offrono completa cSopravvivenza del genoma. In un recente studio del nostro laboratorio 7 , sono stati utilizzati metodi basati su immunoprecipitazione, piuttosto che la conversione di bisulfite, per identificare regioni ricche di CpG ricche e differenziate su scala globale nei pazienti CLL e nei controlli normali sani. Il sequenziamento di prossima generazione (MBD-seq) del dominio di binding inmetil-CpG (MBD), l&#39;arricchimento del DNA frammentato a doppio filamento dipende dal grado di metilazione di CpG. Questo metodo può superare gli inconvenienti del metodo di conversione di bisulfite e può anche fornire una copertura genomica della metilazione CpG in modo indipendente e indipendente dalla PCR. Inoltre, a differenza dei metodi di microarray basati su conversione a base di bisolfito, MBD-seq può essere utilizzato per analizzare lo stato di metilazione degli elementi ripetitivi, quali elementi nucleari interrati lunghi (LINEs), elementi nucleari interspersati corti (SINEs), ripetizioni terminali lunghe (LTRS) Ecc . Tuttavia, rispetto ai metodi di conversione di bisolfito,Un protocollo MBD-seq richiede una quantità relativamente elevata di DNA di input. Inoltre, la qualità dei sequenziamenti legge e i dati dipendono dalla specificità, affinità e qualità degli anticorpi utilizzati. Lo studio corrente spiega un dettagliato protocollo MBD-seq per arricchire il DNA metilato per la sequenza di nuova generazione. Utilizza un kit di arricchimento di legame metilato commerciale (elencato nella tabella dei materiali ), nonché una pipeline computazionale per visualizzare e interpretare i dati di sequenziamento della metilazione per identificare le regioni iper- e ipometilate specifiche CLL rispetto ai controlli normali sani. Fondamentalmente, questo metodo utilizza la capacità dell&#39;MBD dell&#39;interazione con proteine ​​umane MBD2 con CpG metilati per estrarre il DNA arricchito con CpG metilati, e questo è seguito dal sequenziamento ad alta percentuale di DNA metilato.

<table border="0" cellpadding="0" cellspacing="0" width="626">
	<tbody>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nano drop 2000</td>
			<td style="width:124px;">Thermo Fischersceintific</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="51">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vortex mixer</td>
			<td style="width:124px;">VWR</td>
			<td style="width:119px;">12620-848</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Absolute Ethanol</td>
			<td style="width:124px;">Any company</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70% Ethanool</td>
			<td style="width:124px;">Any company</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNAse free water</td>
			<td style="width:124px;">Milli Q</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<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>
		<tr height="42">
			<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>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<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>
			<td style="width:119px;"></td>
			<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>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

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

Il ruolo di lunghi RNA non codificanti (lncRNA) nel cancro sta arrivando all&#39;avanguardia a causa del crescente interesse per comprendere le loro funzioni meccaniche durante lo sviluppo e la progressione del cancro. Nonostante questo, la regolazione epigenetica globale dei lncRNA e delle sequenze ripetitive nei tumori non è stata ben studiata, in particolare nella leucemia linfocitaria cronica (CLL). Questo studio si concentra su un approccio unico: la cattura a base di immunoprecipitazione di frammenti di DNA a doppio filamento e metilato usando proteine ​​del dominio di legame di metile (MBD), seguito da sequenziamento di nuova generazione (MBD-seq). In questo studio sono stati utilizzati campioni di pazienti CLL appartenenti a due sottogruppi prognostici (5 campioni mutati IGVH + 5 IGVH campioni non mutati). L&#39;analisi ha rivelato 5.800 ipermetilati e 12.570 glicini differenzialmente metilati specifici CLL-ipometilati (cllDMGs) rispetto ai normali controlli sani. Importante, questi risultati hanno identificato diversi lncRNA specifici CLL, differenzialmente metilatiElementi petitive e geni che codificano la proteina con potenziale valore prognostico. Questo lavoro descrive un protocollo dettagliato per una pipeline MBD-seq e bioinformatica sviluppata per l&#39;analisi completa dei profili di metilazione globale in regioni altamente ricche di CpG usando campioni di pazienti CLL. Infine, un gene codificante proteina e un lncRNA sono stati convalidati usando il pirosequencing, che è un metodo altamente quantitativo per analizzare i livelli di metilazione di CpG per corroborare ulteriormente i risultati del protocollo MBD-seq.

MBD-seq è stato recentemente eseguito su pazienti affetti da CLL e controlli abbinati, normali e sani per identificare geni differenziali iper- e ipometilati specifici CLL 7 . La pipeline sperimentale e bioinformatica utilizzata per analizzare i dati generati da CLL e campioni sani normali sono mostrati in Figura 1A e 1B . Queste analisi identificarono diverse regioni differenzialmente metilate specifiche CLL (cllDMR...

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

Questo lavoro descrive un protocollo di sequenziamento del dominio di binding di metil-CpG (MBD) ottimizzato e una pipeline computazionale per identificare le regioni ricche di CpG riccamente differenziate in pazienti con leucemia linfocitaria cronica (CLL).

Analisi completa della metilazione del DNA usando un metodo basato sulla cattura di dominio di metile-CpG in pazienti con leucemia linfocitica cronica

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

Determinazione dei percorsi cromosomici ottimali per un elemento di DNA in Escherichia coli usando un approccio novello Transposon-mediata

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

Ricerca

Genetica

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

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

Tempo di replicazione del DNA utilizzando Zebrafish come un sistema di modello In Vivo di profilatura

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

Gene candidato test negli studi clinici di coorte con genotipizzazione multiplex e spettrometria di massa

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

Metodologia per la rilevazione accurata di metilazione del DNA mitocondriale

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

Analisi quantitativa di impionbatura alternativo Pre-mRNA nelle sezioni del cervello del Mouse usando analisi di RNA In Situ di ibridazione

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

Metodi di trasferimento di energia di risonanza a singola molecola per l'analisi in tempo reale della risoluzione dello svincolo di Holliday da 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

Indagine sul ruolo trascrizionale di un silenziatore intronico RUNX1 di CRISPR/Cas9 Ribonucleoprotein acuta

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

Uso dell'analogo della pirimidina, 5-iodo-2′-deossiuridina (IdU) con marcatori del ciclo cellulare per stabilire fasi del ciclo cellulare in una piattaforma di citometria di massa

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

Analisi della metilazione LINE-1 nelle cellule staminali mesenchietratta con Vescicoli Extracellulari Derivati dall'Osteosarcoma

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

Analisi a livello di genoma della distribuzione delle modificazioni isoniche utilizzando il metodo di sequenziamento dell'immunoprecipitazione della cromatina in Magnaporthe oryzae

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