Desideriamo ringraziare i membri della Brown e laboratori Lam per la loro partecipazione criticare questo video e articolo. Questo lavoro è stato sostenuto dai fondi degli Istituti canadesi di ricerca sulla salute e la Michael Smith Fondazione per la Ricerca Sanitaria.

DNA ESTRAZIONE E PREPARAZIONE DEI CAMPIONI DNA da una varietà di diversi campioni (cellule in coltura, fresco congelato così come i tessuti fissati in formalina paraffina) può essere utilizzato per MeDIP. E &#39;importante utilizzare il DNA altamente purificato, senza proteine ​​associate come gli istoni. E &#39;anche importante rimuovere l&#39;RNA il più possibile dal campione, in quanto può interferire sia con la quantificazione del DNA e legame degli anticorpi. La quantità di DNA utilizzata per MeDIP può variare da 200 ng a 1 mg a seconda della quantità di DNA disponibile. Per dimostrare questo protocollo, 1 mg di DNA verrà utilizzato. Il seguente protocollo fornirà alta qualità DNA a doppia elica da cellule in coltura. Altri protocolli dovrebbero essere impiegati per l&#39;estrazione del DNA altri tipi di campioni. <ol><li> Aggiungere 400 ml di buffer di digestione di un pellet di cellule in una provetta da 1,7 ml Eppendorf. </li><li> Aggiungere 100 mg di proteinasi K al tubo, e incubare una notte a 50 ° C. </li><li> Aggiungere 500 microlitri fenolo pH 7 e mescolare delicatamente, ma completamente, agitando. </li><li> Girano a 13 000 g per 10 minuti a temperatura ambiente. </li><li> Rimuovere acquosa (in alto) frazione in una nuova provetta. </li><li> Ripetere i passaggi da 3 a 5 una volta. </li><li> Aggiungere 500 microlitri 01:01 pH fenolo / cloroformio 7 e mescolare delicatamente, ma completamente, agitando. </li><li> Girano a 13 000 g per 10 minuti a temperatura ambiente. </li><li> Rimuovere acquosa (in alto) frazione in una nuova provetta. </li><li> Aggiungere 40 mg di RNasi A e incubare 1 ora a 37 ° C. </li><li> Aggiungere 500 microlitri fenolo pH 7 e mescolare delicatamente, ma completamente, agitando. </li><li> Girano a 13 000 g per 10 minuti a temperatura ambiente. </li><li> Rimuovere acquosa (in alto) frazione in una nuova provetta. </li><li> Ripetere i passaggi da 11 a 13 una volta. </li><li> Aggiungere 500 microlitri 01:01 pH fenolo / cloroformio 7 e mescolare delicatamente, ma completamente, agitando. </li><li> Girano a 13 000 g per 10 minuti a temperatura ambiente. </li><li> Rimuovere acquosa (in alto) frazione in una nuova provetta. </li><li> Aggiunta del volume 1/10th 3 acetato di sodio M (40 mL) e mescolare bene. </li><li> Aggiungere 2 volumi (900 microlitri) del 100% di etanolo, mescolare bene, e posto a -20 ° C per 20 minuti. </li><li> Girano a 13 000 g per 20 minuti a 4 ° C. </li><li> Eliminare l&#39;etanolo, rotazione del polso, ed eliminare l&#39;etanolo residuo. </li><li> Aggiungere 500 microlitri di etanolo freddo al 70% da lavare. Girano a 13 000 g per 20 minuti a 4 ° C. </li><li> Rimuovere etanolo al 70%, rotazione del polso, ed eliminare l&#39;etanolo residuo pipettando. </li><li> Aria secca il pellet con il tappo aperto per 10 minuti a temperatura ambiente per rimuovere ogni traccia di etanolo residuo. </li><li> DNA Risospendere in 50 ml di dH2O sterilizzato notte a 4 ° C. </li><li> Quantificare il DNA utilizzando uno spettrofotometro NanoDrop. Un A 260: A 280 rapporto di 1,8 è l&#39;ideale. </li><li> Determinare la qualità e le dimensioni del DNA su un gel di agarosio con 100 bp scaletta. Meno di 10 ng di DNA genomico può essere eseguito su un 1,7% gel seguita dalla colorazione con un colorante che è molto sensibile a piccole quantità di DNA come oro SYBR. </li><li> In un tubo siliconato per campione, preparare 1 mg di DNA in un volume totale di 50 microlitri con il resto del volume composto sterilizzato con dH 2 O. </li></ol> DNA sonicazione Sonicazione del DNA e il protocollo MeDIP deve essere effettuata in tubi siliconzied a prevenire le non legame specifico delle proteine ​​alle pareti del tubo. Tempi di sonicazione ottimale per i campioni di DNA sono in base al grado di frammentazione del DNA campione determinato da elettroforesi su gel (Punto 27). Per esempio, il DNA estratto dalle cellule in coltura dovrebbero essere di altissimo peso molecolare, e dovranno successivamente essere più sonicazione di DNA estratto da campioni di archivio che sono spesso parzialmente degradate. Se i campioni sono uniformemente alto peso molecolare, è ragionevole a questo punto per procedere con la sonicazione, come descritto in questo protocollo senza controllare ogni campione singolarmente. Se i campioni sono frammentati, come con i campioni d&#39;archivio, sarà necessario adeguare la procedura di sonicazione probabilmente diminuendo i tempi sonicazione di ottenere 300-100bp frammenti. Se si prevede di trattare i campioni che sono parzialmente degradate, l&#39;ottimizzazione dei parametri di sonicazione può essere eseguita su un campione rappresentativo di quelli di interesse. Sulla base del grado di frammentazione del DNA osservata da elettroforesi su gel (Step 27), lo sperimentatore può predeterminare il momento ottimale per sonicazione del campione. Qui si descrive un metodo per ottenere frammenti di DNA 300-1000 bp mediante ultrasuoni con un dispositivo automatico sonicating (Bioruptor da Diagenode, UCD-200 TM) con alto peso molecolare del DNA. <ol><li> Acqua in Biorupter deve essere a 4 ° C. </li><li> Ultrasuoni per 7 minuti a impostazioni automatiche (30 sec a 30 sec fino alla potenza massima). </li><li> Rimuovere 800 ng (40 microlitri) di prodotto sonicato e mettere in provetta da centrifuga siliconato 1,7 ml per la immunoprecipitazione (IP) di reazione. </li><li> Mettere da parte restanti 200 ng (10 microlitri) per servire come input (IN) del DNA di riferimento (conservare a 4 ° C). </li&gt; </ol> IMMUNOPRECIPITATON di DNA metilato <ol><li> Denaturare il DNA che verrà utilizzato per la reazione IP (800 ng) a 95 ° C per 10 minuti in un bagno d&#39;acqua. </li><li> Raffreddare immediatamente in ghiaccio. Lasciate raffreddare completamente il DNA (circa 5 minuti in ghiaccio) prima di procedere con il passo successivo. </li><li> Aggiungere 5 anticorpo monoclonale mcg. </li><li> Aggiungi tampone IP (usato a temperatura ambiente per tutto questo protocollo) per un volume finale di 500 microlitri. </li><li> Incubare per 2 ore a 4 ° C in rotazione titolare tubo. </li><li> Poco prima fase 5 è completa, preparare Dynabeads di lavaggio (Piazza di 6-11). In primo luogo, risospendere le sfere completamente nel flacone con il vortex. </li><li> Trasferire il 30 microlitri (~ 2 x 10 7) di Dynabeads risospeso per reazione più 1, in un nuovo tubo siliconato (per esempio, se facendo 8 reazioni, rimuovere numero sufficiente di sfere per 9, cioè 270 microlitri). </li><li> Posizionare il tubo sul sostegno magnetico per 2 minuti a temperatura ambiente. </li><li> Pipetta il sopranatante. Quando si rimuove il surnatante, evitare di toccare le perline contro parete interna (dove le perle attrarre verso il magnete) con la punta della pipetta. </li><li> Togliere la provetta dal magnete, e risospendere le sfere in un volume in eccesso di tampone IP (750 microlitri-1000 microlitri). Posizionare il tubo di nuovo sul sostegno magnetico per 2 minuti a temperatura ambiente. </li><li> Ripetere il lavaggio una volta di più, e poi risospendere le sfere lavato in tampone IP nel volume originale rimossa al punto 7. </li><li> Aggiungere 30 ml di Dynabeads lavata per ogni reazione IP </li><li> Incubare in un tubo di supporto rotante per 2 ore a 4 ° C. </li><li> Dopo l&#39;incubazione è completa, posizionare il tubo sul sostegno magnetico per 2 minuti a temperatura ambiente. </li><li> Pipetta il sopranatante. Evitare di toccare la parete interna del tubo (dove le perle attrarre verso il magnete) con la punta della pipetta. Aggiungere 500 ml di tampone IP. Mescolare il contenuto della provetta e rimetterlo sul sostegno magnetico per 2 minuti. Ripetere il lavaggio con 500 IP Buffer ul una sola volta. </li><li> Dopo aver rimosso il sopranatante l&#39;ultimo lavaggio, risospendere le sfere in 400 ml di buffer di digestione. </li><li> Trattare la reazione con 100 mg di proteinasi K e incubare una notte a 50 ° C. </li></ol> Purificazione del DNA immunoprecipitato <ol><li> Aggiungere 500 microlitri 01:01 pH fenolo / cloroformio 7 e vortice accuratamente. </li><li> Girano a 13 000 g per 10 minuti a temperatura ambiente. </li><li> Rimuovere acquosa (in alto) frazione in una nuova provetta. </li><li> Ripetere i passaggi da 1 a 3 se l&#39;interfase tra gli strati acquosi e organici aspetto torbido. </li><li> Aggiungere 1 / 10 ° volume di 3 M di acetato di sodio (40 microlitri) e vortice. </li><li> Aggiungere 1 ml di glicogeno (20 mg / mL) e vortice. </li><li> Aggiungere 2 volumi (1000 ml) del 100% di etanolo, vortice, e posto a -20 ° C per 20 minuti. </li><li> Girano a 13 000 g per 20 minuti a 4 ° C. </li><li> Eliminare l&#39;etanolo, rotazione del polso, ed eliminare l&#39;etanolo residuo. </li><li> Aggiungere 500 microlitri di etanolo freddo al 70% da lavare. Vortex brevemente, e far girare a 13 000 g per 20 minuti a 4 ° C. </li><li> Rimuovere etanolo al 70%, rotazione del polso, ed eliminare l&#39;etanolo residuo pipettando. </li><li> Aria secca il pellet con il tappo aperto per 10 minuti a temperatura ambiente per rimuovere ogni traccia di etanolo residuo. </li><li> Risospendere DNA pellet in 10 microlitri sterilizzati dH 2 0. </li></ol> Convalida da parte della PCR <ol><li> Si può verificare per assicurarsi che la procedura sta funzionando MeDIP eseguendo il protocollo MeDIP utilizzando normale DNA umano (maschio o femmina), e successivamente analizzando una regione nota per essere arricchito per metilazione. </li><li> Rimuovere il 30% del prodotto MeDIP al tubo PCR, e un altro 30% del prodotto MeDIP ad un altro tubo di PCR. </li><li> Mettete 10 ng di IN DNA in un tubo di PCR, e 10 ng di IN DNA in un altro tubo PCR. Si dovrebbe ora avere quattro reazioni separate PCR da configurare. </li><li> Eseguire la PCR con H19 e CTRL primer come indicato nella Tabella 1. </li><li> Preparare due mastermixes (uno per ogni set di primer) per le reazioni di PCR con 12,5 microlitri di volume totale come indicato nella Tabella 2. </li><li> Thermocycle la PCR utilizzando le condizioni indicate nella Tabella 3. </li><li> Esegui 5 ml di prodotti di PCR su un gel di agarosio al 2% per la visualizzazione. </li><li> I risultati attesi per MeDIP successo sono riportati nella tabella 4. </li></ol> Tabelle Tabella 1: H19 e CTRL primer per la validazione della PCR. <table border="1"><tbody><tr><td> Set di primer </td><td> Primer avanti </td><td> Primer reverse </td><td> Formato del prodotto atteso </td></tr><tr><td> H19 </td><td> H19_F 5&#39;-cgagtgtgcgtgagtgtgag </td><td> H19_R 5&#39;-ggcgtaatggaatgcttgaa </td><td> 174 bp </td></tr><tr><td> CTRL (controllo) </td><td> CTRL_F5&#39;-gagagcattagggcagacaaa </td><td> CTRL_R 5&#39;-gttcctcagacagccacattt </td><td> 139 bp </td></tr></tbody></table> Tabella 2. Mastermixes per le reazioni PCR. <table border="1"><tbody><tr><td></td><td> H19 Mix (per RXN) </td><td> CTRL Mix (per RXN) </td></tr><tr><td&gt; DDH 2 O </td><td> 6,875 </td><td> 6,875 </td></tr><tr><td> Buffer 10X </td><td> 1,25 </td><td> 1,25 </td></tr><tr><td> dNTP mix (10 mm ciascuno) </td><td> 0,25 </td><td> 0,25 </td></tr><tr><td> MgCl 2 (50 mM) </td><td> 0,25 </td><td> 0,25 </td></tr><tr><td> Primer (H19_F / R o CTRL_F / R) (10 mM ciascuno F / R) </td><td> 0,625 </td><td> 0,625 </td></tr><tr><td> Platinum Taq </td><td> 0,25 </td><td> 0,25 </td></tr></tbody></table> Tabella 3. PCR thermocyling condizioni. <table border="1"><tbody><tr><td></td><td> 95 ° C </td><td> 05:00 </td></tr><tr><td rowspan="3"> 40X </td><td> 95 ° C </td><td> 00:30 </td></tr><tr><td> 56 ° C </td><td> 00:30 </td></tr><tr><td> 72 ° C </td><td> 00:15 </td></tr></tbody></table> Tabella 4. Risultati attesi PCR per MeDIP successo. <table border="1"><tbody><tr><td></td><td colspan="2" align="center"> Modello di DNA </td></tr><tr><td> Primer utilizzati </td><td> IN </td><td> IP </td></tr><tr><td> H19 </td><td> Positivo </td><td> Positivo </td></tr><tr><td> CTRL </td><td> Positivo </td><td> Negativo </td></tr></tbody></table>

<ol>
	<li>Beck, S., Rakyan, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+methylome:+approaches+for+global+DNA+methylation+profiling.">The methylome: approaches for global DNA methylation profiling.</a> Trends Genet. 24, 231-237 (2008).</li><li>Lu, Q., 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=Epigenetics,+disease,+and+therapeutic+interventions.">Epigenetics, disease, and therapeutic interventions.</a> Ageing research reviews. 5, 449-467 (2006).</li><li>Zilberman, D., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+DNA+methylation+patterns.">Genome-wide analysis of DNA methylation patterns.</a> Development. 134, 3959-3965 (2007).</li><li>Feinberg, A. P., Tycko, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+history+of+cancer+epigenetics.">The history of cancer epigenetics.</a> Nature reviews. 4, 143-153 (2004).</li><li>Weber, 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=Distribution,+silencing+potential+and+evolutionary+impact+of+promoter+DNA+methylation+in+the+human+genome.">Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome.</a> Nat Genet. 39, 457-466 (2007).</li><li>Weber, 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=Chromosome-wide+and+promoter-specific+analyses+identify+sites+of+differential+DNA+methylation+in+normal+and+transformed+human+cells.">Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells.</a> Nat Genet. 37, 853-862 (2005).</li><li>Wilson, I. 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=Epigenomics:+mapping+the+methylome.">Epigenomics: mapping the methylome.</a> Cell Cycle. 5, 155-158 (2006).</li><li>Gazin, C., Wajapeyee, N., Gobeil, S., Virbasius, C. M., Green, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+elaborate+pathway+required+for+Ras-mediated+epigenetic+silencing.">An elaborate pathway required for Ras-mediated epigenetic silencing.</a> Nature. 449, 1073-1077 (2007).</li><li>Karpinski, P., Sasiadek, M. M., Blin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+epigenetic+patterns+in+the+etiology+of+gastrointestinal+cancers.">Aberrant epigenetic patterns in the etiology of gastrointestinal cancers.</a> Journal of applied. 49, 1-10 (2008).</li><li>Maekawa, M., Watanabe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics:+relations+to+disease+and+laboratory+findings.">Epigenetics: relations to disease and laboratory findings.</a> Current medicinal chemistry. 14, 2642-2653 (2007).</li><li>Vucic, E. A., Brown, C. J., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics+of+cancer+progression.">Epigenetics of cancer progression.</a> Pharmacogenomics. 9, 215-234 (2008).</li><li>Egger, G., Liang, G., Aparicio, A., Jones, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics+in+human+disease+and+prospects+for+epigenetic+therapy.">Epigenetics in human disease and prospects for epigenetic therapy.</a> Nature. 429, 457-463 (2004).</li><li>Jones, P. A., Baylin, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fundamental+role+of+epigenetic+events+in+cancer.">The fundamental role of epigenetic events in cancer.</a> Nat Rev Genet. 3, 415-428 (2002).</li><li>Fraga, M. F., Esteller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation:+a+profile+of+methods+and+applications.">DNA methylation: a profile of methods and applications.</a> Biotechniques. 33, 632-649 (2002).</li></ol>

Vi è una crescente consapevolezza della metilazione del DNA gioca significativo ruolo nella malattia, quindi lo sviluppo di test per misurare questa modifica sono sempre più importanti 3, 12, 13. La tecnica MeDIP è uno strumento suscettibile di screening, sia l&#39;intero genoma e locus specifico livello 6, 7. Questa tecnica fornisce una visione rapida dei livelli di metilazione del DNA utilizzando quantità limitate di partenza del DNA e permette un facile confronto tra le diverse fonti. Applic...

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		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Biorupter sonicator</td>
<td>Tool</td>
<td>Diagenode</td>
<td>UCD-200 TM</td>
<td>&nbsp;</td>
<tr>
<td>1.7ml SafeSeal Microcentrifuge Tubes</td>
<td>Other</td>
<td>Sorenson BioScience</td>
<td>11510</td>
<td>&nbsp;</td>
<tr>
<td>ND 3300 Spectrophotometer</td>
<td>Tool</td>
<td>NanoDrop</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Primary Antibody: Anti-5'-methylcytosine mouse mAb</td>
<td>Reagent</td>
<td>CalBiochem</td>
<td>162 33 D3</td>
<td>&nbsp;</td>
<tr>
<td>Secondary Antibody: Dynabeads M-280 Sheep anti-mouse IgG</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>112-01D</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic Tube Rack</td>
<td>Tool</td>
<td>Invitrogen</td>
<td>CS15000</td>
<td>&nbsp;</td>
<tr>
<td>Mini LabRoller</td>
<td>Tool</td>
<td>Labnet International</td>
<td>H5500</td>
<td>&nbsp;</td>
<tr>
<td>IP Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10 mM NaPO4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100. Stored at room temperature</td>
</tr>
<tr>
<td>Digestion Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10 mM Tris pH8.0, 100 mM EDTA, 0.5% SDS, 50 mM NaCl</td>
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methylated dna immunoprecipitation

L&#39;identificazione di modelli di metilazione del DNA è una procedura comune nello studio dell&#39;epigenetica, come metilazione è conosciuto per avere effetti significativi sulla espressione genica, ed è impegnato con il normale sviluppo così come la malattia<sup&gt; 1-4</sup&gt;. Così, la capacità di discriminare tra il DNA metilato e non metilato del DNA è essenziale per la generazione di profili di metilazione per tali studi. DNA metilato immunoprecipitazione (MeDIP) è una tecnica efficace per l&#39;estrazione del DNA metilato da un campione di interesse<sup&gt; 5-7</sup&gt;. Un campione di appena 200 ng di DNA è sufficiente per l&#39;anticorpo, o immunoprecipitazione (IP), reazione. Il DNA è sonicato in frammenti di dimensioni variabili da 300-1000 bp, ed è diviso in immunoprecipitati (IP) e ingresso (IN) porzioni. DNA IP viene successivamente calore denaturato e poi incubate con anti-5&#39;mC, permettendo l&#39;anticorpo monoclonale di legare il DNA metilato. Dopo questo, sfere magnetiche che contiene un anticorpo secondario con affinità per l&#39;anticorpo primario sono aggiunti, e si incuba. Queste perline legati anticorpi si legano l&#39;anticorpo monoclonale utilizzato nella prima fase. DNA legato al complesso anticorpale (DNA metilato) è separata dal resto del DNA, utilizzando un magnete per tirare i complessi in soluzione. Numerosi lavaggi utilizzando tampone IP vengono poi eseguite per rimuovere l&#39;, non legato non metilato DNA. La metilazione del DNA / anticorpo vengono digeriti con proteinasi K per digerire gli anticorpi lasciando solo il DNA metilato intatto. Il DNA purificato è arricchito da fenolo: estrazione con cloroformio per rimuovere la questione delle proteine ​​e poi precipitare e risospeso in acqua per un uso successivo. Tecniche di PCR può essere utilizzato per convalidare l&#39;efficienza della procedura MeDIP analizzando i prodotti di amplificazione del DNA IN IP e per le regioni noti per mancanza e noti per contenere sequenze metilate. Il DNA purificato metilato può quindi essere utilizzata per locus-specifici (PCR) o genome-wide (microarray e sequenziamento) studi di metilazione, ed è particolarmente utile quando applicato in combinazione con altri strumenti di ricerca quali profili di espressione genica e l&#39;ibridazione genomica comparativa serie ( CGH)<sup&gt; 8</sup&gt;. Ulteriori indagini in metilazione del DNA porterà alla scoperta di nuovi bersagli epigenetici, che a sua volta, può essere utile per lo sviluppo di nuovi strumenti di ricerca terapeutica o prognostici per malattie come il cancro che sono caratterizzati da metilazione aberrante del DNA<sup&gt; 2, 4, 9-11</sup&gt;.

Watch this Scientific Journal Video about Methylated DNA Immunoprecipitation at JoVE.com

Methylated DNA Immunoprecipitation

Questo video dimostra il protocollo di immunoprecipitazione DNA metilato (MeDIP). MeDIP è una procedura di due giorni che estrae selettivamente i frammenti di DNA metilato da un campione di DNA genomico utilizzando anticorpi con specificità per 5-metilcitosina (anti-5 MC).

Immunoprecipitazione DNA metilato

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

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Biology

23.2K Views.  BC Cancer Research Centre. Questo video dimostra il protocollo di immunoprecipitazione DNA metilato (MeDIP). MeDIP è una procedura di due giorni che estrae selettivamente i frammenti di DNA metilato da un campione di DNA genomico utilizzando anticorpi con specificità per 5-metilcitosina (anti-5 MC).

Video: Methylated DNA Immunoprecipitation

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Immunoprecipitazione della cromatina di cellule staminali embrionali umane

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

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Biologia

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Profiling anticorpi Systems Immunoprecipitazione luciferasi (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

DNA Stable-Isotope Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

DNA Stable-Isotope Probing DNA-SIP

DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.

DNA Stabile-isotopica Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and ...

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP-FCM: Immunoprecipitazione rilevato mediante citometria di flusso

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Saggio di immunoprecipitazione della cromatina per geni tessuto-specifici utilizzando nella fase iniziale di embrioni di topo

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

Immunoprecipitazione della cromatina (ChIP) con Drosophila Tessuto

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters within the origin of replication1. Beyond the origin, SeqA is found at the replication forks, where it organizes newly replicated DNA into higher ordered structures2. SeqA associates only weakly with single GATC sequences, but it forms high affinity complexes with DNA duplexes containing multiple GATC sites. The minimal functional and structural unit of SeqA is a dimer, thereby explaining the requirement of at least two GATC sequences to form a high-affinity complex with hemimethylated DNA3. Additionally, the SeqA architecture, with the oligomerization and DNA-binding domains separated by a flexible linker, allows binding to GATC repeats separated by up to three helical turns. Therefore, understanding the function of SeqA at a molecular level requires the structural analysis of SeqA bound to multiple GATC sequences. In protein-DNA crystallization, DNA can have none to an exceptional effect on the packing interactions depending on the relative sizes and architecture of the protein and the DNA. If the protein is larger than the DNA or footprints most of the DNA, the crystal packing is primarily mediated by protein-protein interactions. Conversely, when the protein is the same size or smaller than the DNA or it only covers a fraction of the DNA, DNA-DNA and DNA-protein interactions dominate crystal packing. Therefore, crystallization of protein-DNA complexes requires the systematic screening of DNA length4 and DNA ends (blunt or overhang)5-7. In this report, we describe how to design, optimize, purify and crystallize hemimethylated DNA duplexes containing tandem GATC repeats in complex with a dimeric variant of SeqA (SeqA&Delta;(41-59)-A25R) to obtain crystals suitable for structure determination.

Crystal structure of protein&#x2013;DNA complexes can provide insight into protein function, mechanism, as well as, the nature of the specific interaction. Here, we report how to optimize the length, sequence and ends of duplex DNA for co-crystallization with Escherichia coli SeqA, a negative regulator of replication initiation.

Ottimizzazione iterativa di duplex di DNA per cristallizzazione di Seqa-DNA Complessi

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters ...

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

Efficiente immunoprecipitazione della cromatina utilizzando Limitare quantità di biomassa

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

Generazione di immunoprecipitazione della cromatina Template DNA di alta qualità per high-throughput sequencing (ChIP-Seq)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

In tempo reale Imaging del singolo Engineered RNA trascritti in cellule viventi Utilizzando Ratiometric bimolecolari Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...