L&#39;assistenza tecnica e la strumentazione della University of Maryland Greenebaum Cancer Center traslazionale Nucleo Fondo, in particolare Drs. Rena Lapidus e Mariola Sadowska, sono riconosciuti con gratitudine. Il lavoro di responsabile per lo sviluppo di questo test è stato finanziato in parte dal NIH RO1CA108846, AHA Grant-in-Aid GRNT2130014, un VA Merit Award per AP, e l&#39;Università del Maryland sigaretta restituzione Funds (CRF) fornita al Marlene &amp; Stewart Greenebaum Cancer Center.

Diverse fasi vengono eseguite in anticipo e diversi reagenti vengono preparate e conservate prima della procedura: (1) cultura e proteine ​​cella di isolamento, (2) preparazione di oligonucleotide, (3) Preparazione di piastre a 96 pozzetti, (4) estratto nucleare notte di incubazione. Procedura prevede due giorni a causa della notte di incubazione della proteina nucleare con oligonucleotidi di DNA. 1. Preparazione del buffer 1.1 isolamento delle proteine ​​nucleari <ol><li> Ipotonica Buffer: Per preparare 100 ml di tampone ipotonico, aggiungere 1 ml (1 M HEPES, pH 7.9), 150 ml (1 M MgCl 2), 333 ml (3 M KCl) a 98,3 ml H 2 O. Concentrazioni finali: HEPES 10 mM, 1,5 mM MgCl 2, KCl 10 mM. Per tampone ipotonico + NP40 (per l&#39;utilizzo in fase 2.11): Aggiungere 0,5 ml (10% NP40) per 9,5 ml di tampone ipotonico per la finale dello 0,5% NP40. </li><li> Salt Buffer Bassa: Per preparare 100 ml di tampone sale basso (per l&#39;utilizzo in fase 2.15), aggiungere 1 ml (1 M HEPES, pH 7.9), 25 ml glicerolo, 150 ml (1 M MgCl 2), 666 ml (3 M KCl), 40 ml (0,5 M EDTA) a 73 ml di H 2 O. Le concentrazioni finali: HEPES 10 mM, 25% glicerolo, 1,5 mM MgCl 2, 20 mM KCl, 0,2 mM EDTA. </li><li> Buffer di sale: Per preparare 100 ml di tampone sale alto (per l&#39;utilizzo in fase 2.15), aggiungere 1 ml (1 M HEPES, pH 7.9), 25 ml di glicerina, 150 ml (1 M MgCl 2), 26,7 ml (3 M KCl), 40 microlitri (0.5M EDTA) a 47 ml di H 2 O. Concentrazioni finali: HEPES 10 mM, 25% glicerolo, 1,5 mM MgCl 2, 800 mM KCl, 0,2 mM EDTA. </li><li> 10% NP40 detergente: 10 ml NP40 a 90 ml H 2 O </li><li> Gli inibitori della proteasi e fosfatasi [aggiunto al buffer ipotonico + NP40 (passo 2.11), al buffer basso contenuto di sale (punto 2.15), e al buffer di sale (punto 2.15) appena prima dell&#39;uso]: Aggiungere 2,5 microlitri (1 M DTT) e 50 microlitri di inibitori delle proteasi (100x) a 5 ml di tampone per ciascuna concentrazione finale di 0,5 mM DTT e inibitori della proteasi 1x. Il 100x magazzino miscela di inibitori della proteasi comprende:fluoruro di sodio (Ser / Thr, fosfatasi acida), orthovanadate sodio (Tyr, fosfatasi alcalina), β-glicerofosfato (fosfatasi Ser / Thr), pirofosfato di sodio (fosfatasi Ser / Thr), aprotinina (Servizi proteasi), Bestatin (amino-peptidasi ), E64 (proteasi cisteina), leupeptina (proteasi Ser / Cys), EDTA (metalloproteasi). </li></ol> 1.2 Preparazione delle piastre di saggio <ol><li> Piatto soluzione di fissaggio: Per preparare 500 ml di soluzione, aggiungere 5,25 g di carbonato di sodio per 500 ml H 2 O, mescolare bene e aggiustare il pH a 9,7 per una concentrazione finale di 0,1 M di carbonato di sodio. </li><li> Piatto soluzione di blocco: Per preparare magazzino poli dI / dC oligonucleotide, risospendere 1 Unit (~ 50 mg) di poli dI / DC in 1 ml di 10 mM Tris / HCl, pH 7,9 contenente 1 mM EDTA per una concentrazione di scorta di 50 mg / microlitri. Stock è diluita a 1 mg / mL prima dell&#39;uso. </li></ol> 1.3 tamponi di lavaggio e diluizioni di anticorpi NOTA: tampone di lavaggio Streptavidin è<html> utilizzato per lavare piatti tra aggiunte e per diluire anticorpi primari e secondari. <ol><li> Per preparare 1 L di tampone di lavaggio Streptavidin, aggiungere 2,4 g Tris / HCl, 37,5 ml (4 M NaCl), 10 ml (10% BSA), 5 ml (10% Tween-20), a 1 L di acqua filtrata Millipore sterile , pH 7.18. Conservare a 4 ° C. Le concentrazioni finali: 20 mM Tris / HCl, 150 mM NaCl, 0,1% BSA, 0,05% Tween-20. </li></ol> 1.4 tampone di legame al DNA NOTA: Questo tampone è conservato a -20 ° C. <ol><li> Per preparare 15 ml di DNA binding buffer, aggiungere 180 microlitri (1 M HEPES, pH 7.9), 300 ml (3 M KCl), 12 ml (0.5 M EDTA, pH 8.0), 7.5 ml (1 M DTT), 3,0 ml (60% glicerolo), 300 ml (50 mg / ml poly dI / dC) diluito con acqua deionizzata / distillata H 2 O (12 ml). Concentrazioni finali: 12 mM HEPES / pH 7.9, 60 mM KCl, 0,4 mM EDTA, DTT 0,5 mm, 12% glicerolo, 1 mg / ml poly dI / DC. </li></ol> 2. Coltura cellulare e isolamento proteina nucleare t &quot;&gt; NOTA: La preparazione richiede circa 3 ore La stimolazione di cellule dipenderà esperimento Il numero di cellule utilizzate per ciascun esperimento varierà e tutti i volumi riflette un tipico esperimento utilizzando 3 x 10 7 cellule / punto Regolare volumi per accogliere... il numero di cellule effettivamente utilizzato nell&#39;esperimento 6. <ol><li> Per preparare proteina nucleare di DNA saggi di legame, le cellule umane cultura che esprimono RUNX2 (endoteliali, osteosarcoma, le cellule del cancro al seno) nelle matrici appropriate 6. </li><li> Trattare le cellule subconfluenti con nocodazole (0,2 mg / ml per 16 ore) per arrestare le cellule al confine ciclo cellulare G2 / M 6. In queste condizioni, la proteina RUNX2 viene stabilizzato e vincolante DNA massimali si ottengono. In questo modo, proteina nucleare sufficiente è disponibile per analisi multiple e inibitori possono essere confrontati dalla stessa preparazione. </li><li> Per ogni passo, pre-raffreddare la centrifuga a 4 ° C e preparare buffer e chill 20 min a 4 ° C prima della raccolta delle cellule.</li><li> Cellule freddo (4 ° C) e le procedure condotta su ghiaccio. </li><li> Centrifugare le cellule a 1000 rpm per 5 minuti in 15 ml di polipropilene tubo tondo a fondo. </li><li> Lavare 1x con PBS freddo (Ca 2 + e Mg 2 + libero). </li><li> Centrifuga di nuovo e scartare PBS surnatante. </li><li> Aggiungere 500 ml di tampone ipotonico senza inibitori della proteasi, facendo attenzione a risospendere completamente il pellet cellulare. </li><li> Trasferire contenuto in una provetta da 1,5 ml Eppendorf. </li><li> Subito centrifugare a 5.500 rpm per 5,5 min; scartare il surnatante e mantenere il pellet di cellule. </li><li> Risospendere il pellet di cellule in 500 microlitri di tampone ipotonico contenente 0,5% NP40. Aggiungi proteasi e gli inibitori della fosfatasi e DTT 0.5 mM (come descritto al punto 1.1.5). </li><li> Lasciare il campione a riposo in ghiaccio per 30 min. </li><li> Centrifugare a 13.000 rpm per 10 min. </li><li> Scartare il surnatante (contiene le proteine ​​citosoliche). </li><li> Risospendere il pellet nucleare in 60 ml ciascuna di bassa saltampone t (per cui gli inibitori della proteasi e DTT 0,5 mM sono stati aggiunti dal punto 1.1.5) e tampone salina (a cui gli inibitori della proteasi e DTT 0,5 mM sono stati aggiunti dal punto 1.1.5) per un totale di 120 microlitri. Aggiungere 60 ml di tampone sale basso per il pellet e risospendere prima, quindi aggiungere 60 ml di tampone di sale. Agitare il pellet per facilitare la risospensione. </li></ol> NOTA: Questo passaggio aiuta a sbarazzarsi di alcune delle proteine ​​di membrana nucleare e predisposto per la fase di lisi: basso contenuto di sale per primo (risospendere pellet, vortex), poi di sale (vortex) ottimizza questo passo. Mantenere estratto in ghiaccio per 30 min, con vortex dopo i primi 10 min. <ol start="16"><li> Centrifugare a 12.000 rpm per 15 min; conservare il surnatante contenente la proteina nucleare. </li><li> Misurare la concentrazione di proteine ​​(cercare di mantenere i campioni a 2-5 mg / ml), aliquota in quantità adeguate (10 ml / tubo) e conservare a -80 ° C. </li></ol> NOTA: rendimento stimato: 30 x 10 6 cells produrranno 5 mg / ml di proteine ​​in 120 microlitri. 5 x 10 6 cellule produrranno 2,2 mg / ml di proteine ​​in 40 microlitri. 3. Preparazione di Double Stranded oligonucleotide <ol><li> Progettare oligonucleotidi complementari con siti metà compatibili alle estremità. Per RUNX2 questi sono: 5&#39;-CGTATT AACCACA ATACTCGCGTATT AACCACA ATACTCGCGTATT AACCACA ATAC TCG-3&#39;-biotina (senso) e 5&#39;CGAGTAT TGTGGTT AATACGCGAGTAT TGTGGTT AATACGCGAGTAT T GTGGTT AATACG-3&#39;-biotina (antisenso). </li></ol> NOTA: esperimenti pilota determinato che tre siti di legame Runx2 e single-end biotina marcata hanno prodotto i risultati più riproducibili. <ol start="2"><li> Preparare buffer di ricottura 5x: Aggiungere 300 ml (1 M Tris-HCl, pH 7.6), 30 ml (1 M MgCl 2), 10 ml (1 M DTT) a 260 ml H 2 O (finale di 600 microlitri). </li><li> Per 200 pmol di ogni olig singolo filamentoonucleotide (3 ml), aggiungere 12 ml di tampone 5x ricottura e 45 ml H 2 O per un totale di 60 microlitri (200 pmol/60 microlitri). </li><li> Riscaldare a 95 ° C per 5-10 minuti su una piastra riscaldante. </li><li> Raffreddare a 65 ° C lentamente (impostando la temperatura a 65 ° C, ci vogliono 15 min), poi raffreddare lentamente a temperatura ambiente spegnendo l&#39;alimentazione del blocco riscaldante (o mettendo in 50 ml di acqua 65 ° C in un becher su ghiaccio; questo richiede 15-20 min). </li></ol> 4. Preparazione di piastre con 96 pozzetti NOTA: non utilizzare proteine ​​del latte nei buffer di lavaggio. <ol><li> Fissare la piastra con la soluzione di fissaggio (0.1 M carbonato di sodio): aggiungere 300 microlitri / pozzetto. </li><li> Incubare per 2 ore agitando a temperatura ambiente (RT), o nessuna dondolo a 4 ° C (O / N). </li><li> Lavare 3x piastra con tampone di lavaggio streptavidina (WB): aggiungere 300 microlitri / bene ogni volta. </li><li> Aggiungi a doppio filamento oligonucleotide marcato con biotina (3 consensus siti di legame per nucleotide) per 2 ore wesimo dondolo (1,25 nmol / e; 100 microlitri / pozzetto). </li><li> Lavare 3x con freddo WB: aggiungere 300 microlitri / bene e poi invertire immediatamente la piastra in un lavandino e macchia i bordi su un tovagliolo di carta dopo ogni aggiunta. </li></ol> NOTA: non utilizzare puntali delle pipette per rimuovere il liquido dalle piastre in quanto ciò potrebbe raschiare avidina: biotina: complessi di DNA dai pozzetti. Fate questo per tutte le fasi di lavaggio successive. 5. Incubazione con estratto nucleare NOTA: Non sostituire salmone o aringa DNA di sperma per la poli tampone di bloccaggio dI / dC. Evitare di bloccare piatti con proteine ​​del latte. Entrambi questi agenti bloccanti come risultato valori di fondo alti. <ol><li> Incubare con fattore di trascrizione specifico elaborato da estratto nucleare (90 microlitri / pozzetto). Preparare una master mix contenente estratto nucleare (DNA binding protein; 3-9 mg / pozzetto) + poly dI / DC (1 mg / mL da un mcg / magazzino ul 50) nel DNA 1x tampone di legame. Volume è come necessario per il numero totale dipozzetti necessari (90 microlitri / pozzetto). </li><li> Qualsiasi inibitore potenziale, come la vitamina D3 (figura 2) o CADD composto 5.221.975 (Figura 3), o la diluizione appropriata di controllo solvente, come etanolo, viene aggiunto in questa fase. </li><li> Luogo piastra su piattaforma oscillante, O / N a 4 ° C. </li><li> Lavare 3x con WB: aggiungere 300 microlitri / pozzetto </li></ol> 6. L&#39;aggiunta di anticorpo primario NOTA: diluizioni anticorpo primario deve essere preparato fresco - lo stoccaggio non è raccomandato. <ol><li> Diluire specifico RUNX2 anticorpo monoclonale (magazzino 1 mg / mL) 1/5, 000 in Streptavidin tampone di lavaggio. Preparare sufficiente per aggiunta a ciascun pozzetto della piastra da 96 pozzetti (90 microlitri / pozzetto) in triplicato. </li><li> Incubare per 1 ora a temperatura ambiente su una piattaforma oscillante. </li><li> Lavare 3x con WB, aggiungere 300 ml / pozzetto. </li></ol> 7. L&#39;aggiunta di anticorpo secondario NOTA: diluizioni di anticorpi secondari possono essere STOrosso a 4 ° C durante la notte se necessario il giorno successivo. <ol><li> Diluire F ab-specifica affinità purificato HRP-anticorpo coniugato (7,1 mg / ml di magazzino) per 1:1,400 in Streptavidin lavare buffer: buffer di 3,5 microlitri antibody/5.0 ml lavaggio. Preparare sufficiente per aggiunta a ciascun pozzetto della piastra da 96 pozzetti (90 microlitri / pozzetto) in triplicato. </li><li> Incubare per 30 minuti a temperatura ambiente su una piattaforma oscillante. </li><li> Lavare 6x con WB invertendo piastra nel lavandino (300 microlitri / pozzetto). </li></ol> 8. HRP substrato e Sviluppo Prodotti <ol><li> Aggiungere 50 ml di substrato TMB per pozzetto direttamente dal magazzino bottiglia. </li><li> Incubare 10-20 min a RT al buio (fermata metodo di reazione) o misurare l&#39;assorbanza direttamente (monitoraggio cinetica continuo). </li></ol> 9. Misura di reazione <ol><li> Fermare metodo della reazione: Per la reazione di arresto visiva, verificare la presenza di cambiamento di colore dal chiaro al blu e smettere di reazione con 50 ml di acido solforico. </li><li> Misurare l&#39;assorbanza a 450 nm conil Biotrak II lettore di piastre visibile Spettrofotometro (Amersham Biosciences, GE Healthcare Biosciences Corp. Piscataway NJ, USA) o uno strumento simile. </li><li> Continuo metodo di monitoraggio cinetico: Aggiungere substrato dopo l&#39;ultimo lavaggio e appena prima messa in spettrofotometro (non fermare la reazione). </li><li> Misurare l&#39;assorbanza a 635 nm con il Bio-Tek Synergy HT Multi-reazione lettore di micropiastre spettrofotometro (Bio-Tek Instruments, Inc; Winooski, VT, USA) o uno strumento simile. </li></ol>

<ol>
	<li>Hellman, L. M., Fried, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+mobility+shift+assay+(EMSA)+for+detecting+protein-nucleic+acid+interactions.">Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions.</a> Nat. Protoc. 2, 1849-1861 (2007).</li><li>Bhattacharya, N., Sarno, A., Idler, I. 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=High-throughput+detection+of+nuclear+factor-kappaB+activity+using+a+sensitive+oligo-based+chemiluminescent+enzyme-linked+immunosorbent+assay.">High-throughput detection of nuclear factor-kappaB activity using a sensitive oligo-based chemiluminescent enzyme-linked immunosorbent assay.</a> Int. J. Cancer. 127, 404-411 (2010).</li><li>Hartzell, D. D., Trinklein, N. D., Mendez, 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=A+functional+analysis+of+the+CREB+signaling+pathway+using+HaloCHIP-chip+and+high+throughput+reporter+assays.">A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high throughput reporter assays.</a> BMC Genomics. 10, 497 (2009).</li><li>Vuori, K. A., Ahlskog, J. K., Sistonen, L., Nikinmaa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TransLISA,+a+novel+quantitative,+nonradioactive+assay+for+transcription+factor+DNA-binding+analyses.">TransLISA, a novel quantitative, nonradioactive assay for transcription factor DNA-binding analyses.</a> FEBS J. 276, 7366-7374 (2009).</li><li>D'Souza, D. R., Salib, M. M., Bennett, 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=Hyperglycemia+Regulates+RUNX2+Activation+and+Cellular+Wound+Healing+through+the+Aldose+Reductase+Polyol+Pathway.">Hyperglycemia Regulates RUNX2 Activation and Cellular Wound Healing through the Aldose Reductase Polyol Pathway.</a> J. Biol. Chem. 284, 17947-17955 (2009).</li><li>Underwood, K. F., D'Souza, D. R., Mochin, M. 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=Regulation+of+RUNX2+transcription+factor-DNA+interactions+and+cell+proliferation+by+Vitamin+D3+(cholecalciferol)+prohormone+activity.">Regulation of RUNX2 transcription factor-DNA interactions and cell proliferation by Vitamin D3 (cholecalciferol) prohormone activity.</a> J. Bone Miner Res. 27, 913-925 (2012).</li><li>Held, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+ß-galactosidase+activity+using+the+PowerWave+HT+and+Gen5+data+analysis+software:+basic+enzyme+kinetic+determinations.">Kinetic analysis of ß-galactosidase activity using the PowerWave HT and Gen5 data analysis software: basic enzyme kinetic determinations.</a> Biotek Instruments Application Note. , (2007).</li><li>Renard, P., Ernest, I., Houbion, A., 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=Development+of+a+sensitive+multi-well+colorimetric+assay+for+active+NFkappaB.">Development of a sensitive multi-well colorimetric assay for active NFkappaB.</a> Nucleic Acids Res. 29, E21 (2001).</li><li>Pommier, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+topoisomerase+I+inhibitors:+chemistry,+biology,+and+interfacial+inhibition.">DNA topoisomerase I inhibitors: chemistry, biology, and interfacial inhibition.</a> Chem Rev. 109, 2894-2902 (2009).</li><li>Pennisi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomics.+ENCODE+project+writes+eulogy+for+junk+DNA.">Genomics. ENCODE project writes eulogy for junk DNA.</a> Science. 337, 1159-1161 (2012).</li></ol>

Non abbiamo nulla da rivelare.

DNA-binding saggi vengono utilizzati per misurare la capacità dei fattori di trascrizione per interagire con il DNA. I saggi di legame per DNA includono spostamento elettroforetico mobilità (EMSA) 1 e immuneprecipitation cromatina (ChIP) saggi basati 3 nonché saggi occupata formati da 96 pozzetti 4 come saggi chemiluminescenti 2. L&#39;EMSA è non-quantitativa e utilizza radioattivo (32 P) oligonucleotidi. Questi sono incubati con proteine ​​nucleari e comple...

DNA-binding saggi hanno utilità nel misurare la capacità dei fattori di trascrizione per interagire con il DNA. I saggi di legame al DNA includono elettroforetici saggi mobility shift (EMSA) che dipendono da oligonucleotidi radioattivi 1 o saggi chemiluminescenza 2. Saggi cromatina immuneprecipitation (chip) basate 3 e saggi che impiegano formati da 96 pozzetti 4 sono state descritte. Tuttavia, l&#39;EMSA è un&#39;analisi non quantitativa che richiede l&#39;uso di oligonucleotidi radiomarcati. Quando le proteine ​​nucleari associano con le specifiche sequenze promotrici nucleotide, complessi vincolanti sono ritardati su gel di poliacrilammide e il fattore di trascrizione specifico possono essere convalidati con un anticorpo &quot;supershift&quot;. Abbiamo sviluppato un saggio di legame al DNA quantitativa utilizzando un formato di immunoassorbimento enzimatico (ELISA-D), che è in grado di misurare l&#39;interazione di RUNX2 con DNA-binding sequenze corrispondenti ad elementi promotori definiti igeni bersaglio n Runx2. Uso di un anticorpo anti-RUNX2 fornisce specificità per il dosaggio e la mancanza di radiotracciante distinguere questo saggio dal tradizionale saggio di spostamento gel 5. Rilevamento di complessi di legame è possibile con l&#39;uso di un anticorpo secondario accoppiato a perossidasi di rafano (HRP), che converte un substrato HRP, tetrametil benzidina (TMB) in un prodotto colorato per l&#39;analisi spettrofotometrica. Il saggio qui riportato può incorporare l&#39;uso di oligonucleotidi di DNA mutati i controlli e può essere utilizzato per il rilevamento di inibitori competitivi e non competitivi di DNA vincolanti. Lo screening di nuovi composti antitumorali è possibile anche con questo test.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the Reagent</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Poly dI/dC</td>
			<td>GE Healthcare, Piscataway, NJ</td>
			<td>US20539-5UN</td>
			<td>1 U ~50mg</td>
		</tr>
		<tr>
			<td>RUNX2 antibody</td>
			<td>MBL International Corp., Woburn, MA</td>
			<td>D130-3</td>
			<td>1 mg/ml</td>
		</tr>
		<tr>
			<td>Fab-specific peroxidase conjugated antibody</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>A9917</td>
			<td>7.1 mg/ml</td>
		</tr>
		<tr>
			<td>TMB Substrate (tetramethyl benzidine)</td>
			<td>EXALPHA Biologicals, Shirley, MA</td>
			<td>X1189S</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>Sodium carbonate</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>57995</td>
			<td>Plate-fixing</td>
		</tr>
		<tr>
			<td>Sulfuric Acid</td>
			<td>VWR, West Chester, PA</td>
			<td>BDH-39922-1</td>
			<td>Stop solution</td>
		</tr>
		<tr>
			<td>Multi-well plates</td>
			<td>Greiner Bio-One, Basel, Switzerland</td>
			<td>655996</td>
			<td>Avidin-coated, black sides</td>
		</tr>
		<tr>
			<td>HALT</td>
			<td>Thermo-Scientific/Pierce, Rockford, IL</td>
			<td>78440</td>
			<td>Protease and phosphatase inhibitors</td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td>Various manufacturers</td>
			<td>Laboratory grade</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Table 1. Reagents</td>
		</tr>
		<tr>
			<td>Spectrophotometer: Biotrak II Visible plate reader</td>
			<td>Amersham Biosciences</td>
			<td>&nbsp;</td>
			<td>For use with stop reaction method</td>
		</tr>
		<tr>
			<td>Spectrophotometer: Bio-Tek Synergy HT Multi-reaction microplate reader</td>
			<td>Bio-Tek Instruments, Inc.</td>
			<td>&nbsp;</td>
			<td>For use with continuous kinetic monitoring</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Table 2. Equipment</td>
		</tr>
	</tbody>
</table>

a quantitative assay to study protein:dna interactions, discover transcriptional regulators of gene expression, and identify novel anti-tumor agents

Molti legano il DNA test come test di spostamento mobilità elettroforetica (EMSA), saggi chemiluminescenti, immunoprecipitazione della cromatina (ChIP) a base di saggi e saggi pozzetti-based sono utilizzati per misurare attività del fattore di trascrizione. Tuttavia, questi saggi sono nonquantitative, mancano di specificità, possono comportare l&#39;uso di oligonucleotidi radiomarcati, e non possono essere adattabile per lo screening di inibitori di legame al DNA. D&#39;altra parte, utilizzando un saggio quantitativo di legame al DNA immunoenzimatico (ELISA-D) Test, dimostriamo interazioni proteina nucleare con DNA utilizzando il fattore di trascrizione RUNX2 che dipendono specifica associazione con consenso DNA-binding sequenze presenti sulla biotina- oligonucleotidi etichettati. Preparazione delle cellule, estrazione di proteine ​​nucleari, e la progettazione di oligonucleotidi a doppio filamento sono descritti. Piastre a 96 pozzetti rivestite di avidina sono fissati con tampone alcalino e incubate con proteine ​​nucleari a nucleotide tampone di bloccaggio. Follgrazie lavaggio estensivo delle piastre, l&#39;anticorpo primario specifico e incubazioni anticorpi secondari sono seguiti dall&#39;aggiunta di substrato perossidasi di rafano e lo sviluppo della reazione colorimetrica. Modalità di reazione di arresto o il monitoraggio continuo cinetica sono stati utilizzati per misurare quantitativamente l&#39;interazione delle proteine ​​con il DNA. Discutiamo adeguati controlli specificità, compreso il trattamento con i non-IgG specifiche o senza proteine ​​o anticorpo primario. Applicazioni del saggio sono descritte compresa la sua utilità in screening di farmaci e risultati positivi e negativi rappresentativi sono discussi.

Il metodo D-ELISA è altamente specifico per la proteina legante il DNA designato finché viene utilizzato un oligonucleotide a doppio filamento sequenza-specifica, contenente tre copie del sito di legame consenso RUNX2 (ACACCA). L&#39;anticorpo primario che riconosce la proteina che migliora anche la specificità. L&#39;anticorpo secondario contiene covalentemente legata perossidasi di rafano (HRP) che converte un substrato trasparente (tetrametil benzidina) per un prodotto colorato per facilità di rilevamento <strong...

Watch this Scientific Journal Video about A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents at JoVE.c

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Abbiamo sviluppato un DNA-binding, dosaggio quantitativo basato su ELISA per misurare le interazioni tra fattori di trascrizione con il DNA. Alta specificità per la proteina RUNX2 è stato raggiunto con un consenso DNA riconoscimento oligonucleotide e anticorpo monoclonale specifico. Rilevamento colorimetrico con una reazione substrato anticorpo enzimatico accoppiato stata monitorata in tempo reale.

Un quantitativo test per studiare proteine: DNA interazioni, Discover trascrizionali regolatori dell&#39;espressione genica, e identificare nuovi agenti anti-tumorali

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

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JoVE Journal

Biology

18.5K Views.  University of Maryland School of Medicine.  Abbiamo sviluppato un DNA-binding, dosaggio quantitativo basato su ELISA per misurare le interazioni tra fattori di trascrizione con il DNA. Alta specificità per la proteina RUNX2 è stato raggiunto con un consenso DNA riconoscimento oligonucleotide e anticorpo monoclonale specifico. Rilevamento colorimetrico con una reazione substrato anticorpo enzimatico accoppiato stata monitorata in tempo reale. 

Video: A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

La preparazione di colture primarie di cellule emopoietiche dal midollo osseo murino per elettroporazione

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

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Biologia

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Utilizzando il sistema di elettroporazione Gene Pulser MXcell per trasfettare cellule primarie con alta efficienza

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Hi-C: un metodo per studiare l&#39;architettura tridimensionale dei genomi.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

L&#39;utilizzo di un contatore automatico cella per semplificare gli studi sull&#39;espressione genica: Knockdown siRNA di IL-4 genica dipendente nelle celle Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

Immunoprecipitazione della cromatina (ChIP) per analisi modifica dinamica Histone nell&#39;espressione genica attivata nelle cellule umane

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Ricostituzione in vitro di Active T. castaneum telomerasi

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolamento dei ribosomi Polipeptidi Bound nascenti<em&gt; In vitro</em&gt; Per identificare i siti di pausa traslazione lungo mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Analisi globale di RNA Sintesi a livello di singola cellula seguente ipossia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA screening per identificare ubiquitina e ubiquitina-come regolatori del sistema di percorsi biologici in cellule di mammifero coltivate

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.