Ringraziamo T. Hung, MC. Tsai, O. Manor, E. Segal, M. Kuroda, T. Swigut e I. Shestopalov per le discussioni. Supportato da parte dell&#39;Agenzia di Scienza, Tecnologia e Ricerca di Singapore (CC), NIH R01-R01-CA118750 e HG004361 (HYC), e la California Institute for Regenerative Medicine (HYC). HYC è uno scienziato inizi della carriera del Howard Hughes Medical Institute.

1. Sonda di progettazione Design anti-senso DNA piastrellatura sonde per il recupero selettivo di RNA bersaglio CHIRP. <ol><li> Progettazione anti-senso sonde oligo con il progettista in linea sonda <a href="http://singlemoleculefish.com">singlemoleculefish.com</a> 18. </li><li> Utilizzare questi parametri: numero di sonde sonda = 1/100 bp di lunghezza RNA, 2) sulla destinazione GC = 45; 3) la durata oligonucleotide = 20; 4) Lunghezza distanza = 60-80. Rompere RNA in segmenti, se troppo a lungo per il progettista. Omettere regioni di ripetizioni o di una ampia omologia. </li><li> Ordina anti-senso sonde di DNA con BiotinTEG a 3-prime. </li><li> Sonde etichetta secondo le loro posizioni lungo l&#39;RNA. Separarli in due gruppi in modo che il pool di &quot;anche&quot; contiene tutte le sonde di numerazione 2, 4, 6, ecc e la piscina &quot;dispari&quot; contiene sonde di numerazione 1, 3, 5, ecc Diluire pool di sonde a 100 micrometri e la concentrazione conservare a -20 ° C. </li><li> Tutti gli esperimenti devono essere eseguite usandoentrambe le piscine, che servono come controlli interni per l&#39;altro. Reale RNA-dipendente segnale sarebbe presente da entrambe le piscine, mentre la sonda specifici rumori sarebbe unico per ogni pool. Questo vale sia per CHIRP-qPCR e CHIRP-seq. </li></ol> 2. Raccogliere cellule Raccogliere cellule che verranno utilizzati per l&#39;esperimento CHIRP. <ol><li> Coltivare cellule in piastre di coltura di tessuto o di palloni confluenza. Risciacquo con tampone fosfato salino (PBS) e una volta Tripsinizzare. Quench tripsina con volume&gt; 2x dei mezzi di comunicazione, pipetta su e giù per staccare le cellule e risospendere in sospensione singola cella. Trasferire tutti i media e le cellule risospese in 50 ml provette Falcon. 20 milioni di cellule sono in genere sufficienti per un campione CHIRP. </li><li> Spin cellule a 800RCF per 4 min. Aspirare il terreno e risospendere 40 milioni di cellule in 40 ml di PBS, unire tubi se necessario. Spin cellule a 800RCF per 4 min. Decantare PBS, aspirare accuratamente in un angolo il liquido rimanente. </li></ol&gt; 3. Cross-Link Cells e raccogliere pellet cellulare Crosslink raccolte le cellule con glutaraldeide per preservare le interazioni RNA-cromatina e preparare pellet di cellule. <ol><li> Eseguire tutti i passaggi a temperatura ambiente. </li><li> Preparare glutaraldeide 1% in PBS temperatura ambiente. Preparare 10 ml per 10 milioni di cellule (0,4 mL magazzino glutaraldeide 25% + 9,6 mL PBS). Glutaraldeide deve essere usato fresco. </li><li> Toccare fondo di tubi Falcon per rimuovere pellets. Risospendere il pellet cellulare in 1% glutaraldeide, a partire da un piccolo volume per evitare blocchi, poi ricaricare fino a tutto volume. Capovolgere. Crosslink per 10 minuti a temperatura ambiente su un end-to-end shaker o dei rotatori. </li><li> Raffreddare bruscamente la reazione di reticolazione con volume 1/10th di 1,25 M glicina a temperatura ambiente per 5 min. </li><li> Spin a 2000RCF per 5 min. Aspirare il surnatante e pellet lavaggio con 20 mL PBS volta raffreddato, filatura a 2000RCF per 5 min. </li><li> Aspirare e risospendere il wincenerito, cross-linked pellet con 1 ml di PBS refrigerata per 20 milioni di cellule. Trasferire ogni ml di una provetta Eppendorf e centrifugare a 2000RCF per 3 min a 4 ° C. Rimuovere il più possibile con PBS punta della pipetta accuratamente. </li><li> Flash-congelare i pellet cellulari in azoto liquido e conservare a -80 ° C a tempo indeterminato. </li></ol> 4. Lisi cellulare Lisare cellule reticolati per preparare lisato cellulare. <ol><li> Scongelare pellet cellulari surgelati a temperatura ambiente. Toccare difficili da rimuovere e miscelare il pellet di cellule. Rallenta il pellet a 2000RCF per 3 min a 4 ° C. Utilizzare una forte punta di 10 microlitri pipetta per rimuovere ogni residuo PBS. </li><li> Su una bilancia elettronica (precisione di 1mg) tarare la massa di un tubo vuoto Eppendorf (i nostri tubi pesano 1.060 grammi molto coerente). Pesare ciascuna pellet e registrare il suo peso. Un piatto completo di 15 cm di cellule HeLa reticolati pesa in genere 100 mg. </li><li> Supplemento Lysis Buffer (10 volte la massa di pellet, ad esempio, 1 ml per 100 mg) con Fresh inibitore della proteasi, PMSF e Superase-in (vedi elenco allegato buffer). Mescolare bene. </li><li> Aggiungi 10X tampone di lisi del volume integrata in ogni provetta e risospendere il pellet. Per le piccole palline &lt;25 mg, risospendere in 250 microlitri di buffer di lisi integrato. Sospensione deve essere liscia. In caso contrario, dividere la sospensione in 500 pl aliquote e utilizzare un mixer motorizzato pellet per rompere grumi. Procedere immediatamente a sonicazione. </li></ol> 5. Sonicazione Shear DNA da lisati cellulari sonicazione reticolati. <ol><li> Sonicare lisato cellulare in Bioruptor in 15 ml provette Falcon. Utilizzare &lt;1,5 ml di lisato in ciascuna provetta, e per una più rapida sonicazione, sonicare non più di due tubi per volta. </li><li> Sottoporre ad ultrasuoni in un bagno di acqua 4 ° C alla massima impostazione con 30 secondi, 45 secondi OFF intervalli di impulso. Controllare lisato ogni 30 min. Continuare sonicazione finché il lisato cellulare è più torbida. Questa operazione potrebbe richiedere un minimo di 30 min e fino a 4 ore. Il numerodi tubi, il volume del campione, la temperatura del bagno, e il periodo di tempo sonicazione che influenzano la durata del processo richiede. Tubi probabilmente ultrasuoni a velocità differenti, in modo da mettere in comune tra loro ogni 30 min e ridistribuire in tubi originali per assicurare l&#39;omogeneità. Nota: glutaraldeide-reticolato cellule richiedere molto più tempo per sonicare equivalenti di formaldeide. </li><li> Quando lisato diventa chiaro, cedere il 5 ul lisato in una nuova provetta Eppendorf. Aggiungere 90 ul DNA proteasi K (PK) Buffer (vedi elenco buffer) e 5 PK ul. Vortex per miscelare e centrifugare brevemente. Incubare per 45 min a 50 ° C. </li><li> Estrarre DNA con il kit di purificazione PCR Qiagen. DNA Eluire in 30 microlitri Buffer Elution Qiagen (EB) e controllare le dimensioni del DNA su gel di agarosio 1%. Se massa della striscio DNA è 100-500 bp, sonicazione è completa. In caso contrario, continuano ad ultrasuoni. </li><li> Centrifugare i campioni sonicati a 16100RCF per 10 minuti a 4 ° C. Combina surnatanti, le aliquote in 1 ml di campioni e flash-congelamento in azoto, liquidon. Conservare a -80 ° C. </li></ol> 6. CHIRP Ibridano sonde di DNA a RNA biotinilati e isolare cromatina legata. <ol><li> Scongelare i tubi della cromatina a temperatura ambiente. </li><li> Preparare il tampone di ibridazione (vedi elenco buffer, preparare 2 ml per ml della cromatina). Vortex per mescolare. </li><li> Per un esempio tipico CHIRP utilizzando 1 ml di lisato, 10 L per rimuovere INPUT RNA e 10 L per INPUT DNA e posto in provette Eppendorf. Tenere in ghiaccio fino al successivo utilizzo. </li><li> Trasferire 1 cromatina mL a 15 mL provetta Falcon. Aggiungere 2 Tampone di ibridazione mL in ciascun tubo. Per volume totale &lt;1,5 ml, utilizzare provette Eppendorf. </li><li> Scongelare sonde a temperatura ambiente. NanoDrop sonde per controllare importo, se non lo avete utilizzato in un tempo lungo (100 sonde pM dovrebbe spec ~ 500-600 ng / ul utilizzando singola impostazione filamento di DNA). Aggiungere il volume appropriato di sonde per tubi specifici (sonda 100 pmol per 1 mL cromatina, 1 ml di 100 pmol / ul sonda per 1 mL cromatina).Mescolare bene. Incubare a 37 ° C per 4 ore con agitazione. </li><li> Con 20 min rimanente per ibridazione, preparare le perline magnetiche C-1 (conservato a 4 ° C). Utilizzare 100 pl per 100 pmol di sonde. Lavare con 1 mL di tampone di lisi senza supplementi per tre volte, utilizzando il DynaMag-2 striscia magnetica di perline separate da buffer. </li><li> Risospendere le sfere in volume originale di tampone di lisi e Supplemento fresca PMSF, PI e Superase-in. Dopo 4 ore reazione di ibridazione è completa, aggiungere 100 microlitri perline in ciascun tubo. Mescolare bene. Incubare a 37 ° C per 30 minuti con agitazione. </li><li> Preparare il tampone di lavaggio (5 mL per campione). Vortex per mescolare. Pre-riscaldare a 37 ° C. Aggiungere PMSF prima dell&#39;uso. </li><li> Lavare perline con 1 mL di tampone di lavaggio cinque volte. Al primo lavaggio, utilizzare DynaMag-15 nastro magnetico a grani separati, decantare, e risospendere in 1 ml di tampone di lavaggio. Trasferire il volume di 1,5 ml provetta Eppendorf. Incubare a 37 ° C con agitazione per 5 min. </li><li> Il lavaggi successivi, spin down ogni tubo su un minicentrifuge, Set di prova sulla DynaMag-2 striscia magnetica per 1 min. Campione di decantare, asciugare eventuali gocce con un Kimwipe, risospendere in 1 ml di tampone di lavaggio. Incubare a 37 ° C con agitazione per 5 min. Ripetere l&#39;operazione per cinque lavaggi totali. </li><li> In ultimo lavaggio, risospendere le sfere bene. Togliere 100 pl e mettere da parte per l&#39;isolamento di RNA. Riserva 900 microlitri per frazione di DNA. Posizionare tutte le provette su DynaMag-2 banda magnetica e rimuovere il tampone di lavaggio. Centrifugare tutte le provette premuto brevemente, metterli su striscia magnetica. Rimuovere l&#39;ultimo bit di tampone di lavaggio completamente con un netto 10 puntale ul. </li></ol> 7. RNA isolamento Estratto frazione di RNA da campioni CHIRP per quantificare da qRT-PCR. <ol><li> Prendete 100 campioni ul tallone e 10 L campione di input RNA. Aggiungere 85 ul RNA PK Buffer pH 7,0 a RNA INPUT. Risospendere le sfere in 95 microlitri di buffer pH 7,0 RNA PK. Aggiungere 5 K ul Proteinease e incubare a 50 ° C per 45 min con end-to-end agitazione. </li><li> In breve lo spin-down tutti i tubi ecampioni bollire per 10 min in blocco di calore a 95 ° C. </li><li> Raffreddare i campioni sul ghiaccio, aggiungere 500 microlitri TRIzol, mescolare vigorosamente nel vortex per 10 sec. Incubare a temperatura ambiente per 10 min. Conservare a -80 ° C o di procedere al punto 4. </li><li> Aggiungere 100 ul al cloroformio Trizol campioni trattati. Energicamente per 10 sec. Spin a 16100RCF su una centrifuga da banco per 15 min a 4 ° C. </li><li> Rimuovere ~ 400 microlitri surnatante acquoso, evitando organico e interfaccia. </li><li> Aggiungere 600 microlitri (1,5 volume) 100% di etanolo e mescolare bene. Spin campione attraverso MIRNeasy colonne mini. Lavare 1x RWT (MIRNeasy mini kit), 2x con RPE al protocollo del produttore. Eluire con 30 microlitri priva di nucleasi H 2 O (NFH 2 O). </li><li> Trattare l&#39;eluato RNA con DNA-free per protocollo del produttore. Dopo la reazione è completa, riscaldare il campione per 15 minuti a 65 ° C per inattivare completamente qualsiasi residuo DNasi. </li><li> Utilizzare 1 RNA ul isolato per bene per qRT-PCR per confermare lncRNArecupero. GAPDH viene spesso utilizzato come controllo negativo. </li></ol> 8. DNA Isolation Estratto frazione di DNA da campioni CHIRP mediante sequenziamento per identificare o quantificare da qPCR. <ol><li> Preparare il tampone di eluizione del DNA (vedi elenco buffer), 150 ml per campione, con ingresso DNA. </li><li> Aggiungere 1 0μL RNasi A (10 mg / ml) e 10 L RNasi H (10 U / pl) per ml di tampone di eluizione DNA, e vortex per miscelare. </li><li> Risospendere ogni campione di perle in 150 microlitri di tampone di eluizione del DNA con RNasi. (DNA INPUT risospendere in 140 pl) incubare a 37 ° C per 30 minuti con agitazione. </li><li> Perle separate e il surnatante DynaMag-2 striscia magnetica. Rimuovere il surnatante e aggiungere ai tubi etichettati. </li><li> Preparare una seconda aliquota di tampone di DNA eluizione con 10 L RNasi A (10 mg / mL) e RNaseH (10 U / mL) esattamente come fatto in 8,2). Aggiungere 150 ul di ogni campione (comprese INPUT DNA), incubare e rimuovere il surnatante. Raccogliere tutte le surnatante (spallad essere ~ 300 mL). </li><li> Aggiungere 15 PK ul a ciascun campione. Incubare a 50 ° C per 45 minuti con agitazione. </li><li> Pre-spin down giallo phase-lock tubi di gel (5PRIME). Trasferimento campioni di DNA di phase-lock tubi di gel, e aggiungere 300 ml PhOH: Cloroformio: isoamile per campione. Agitare vigorosamente per 10 min, e girare su una centrifuga da banco a 16100RCF per 5 min a 4 ° C. Prendere acquoso dalla parte superiore (~ 300 pl). Aggiungere 3 GlycoBlue uL, NaOAc 30 uL, e 900 microlitri EtOH 100%. Mescolare bene e conservare a -20 ° C per una notte. </li><li> Spin campioni a 16100RCF per 30 minuti a 4 ° C. </li><li> Decantare il surnatante con cura. Aggiungere 1 ml di EtOH 70% e vortex per miscelare. Spin a 16100RCF per 5 min. Rimuovere il surnatante con la pipetta. Lasciare asciugare all&#39;aria per 1min. Risospendere in 30 ul EB. </li><li> Campioni di DNA sono pronti per l&#39;analisi mediante qPCR o nella preparazione di high-throughput sequencing librerie per protocollo Illumina. </li></ol> 10. Risultati rappresentativi Figura 1 </strong&gt; descrive il flusso di lavoro CHIRP. Un esperimento CHIRP successo arricchisce tipicamente RNA bersaglio significativamente nel non-specifici RNA. Figura 2 mostra arricchimento della telomerasi RNA umano (TERC) da cellule HeLa oltre GAPDH, un RNA abbondanti cellulari che serve come controllo negativo. La maggior parte degli RNA TERC (~ 88%) presenti nella cellula sono state abbattute eseguendo CHIRP, mentre solo il 0,46% del GAPDH RNA è stato recuperato, dimostrando un fattore di arricchimento di circa 200 volte. Le sonde non specifici quali sonde di targeting LacZ RNA, che non è espressa in cellule di mammifero (Figura 2), può essere usato come addizionali controlli negativi. Regioni di DNA prevede di impegnare la lncRNA destinazione sono in genere arricchiti sulle regioni negativi quando misurata dal qPCR. La figura 3 mostra la convalida qPCR di quattro Hotair-bound siti primari fibroblasti del prepuzio umano che siamo determinati eseguendo CHIRP-seq nella linea stessa cella, mentre TERC e GAPDH DNA siti di sérve come regioni di controllo negativo. Sia &quot;anche&quot; e la sonda &quot;dispari&quot; imposta arricchimento prodotto comparabile attesi Hotair-bound siti su regioni negativi, un segno distintivo di veri lncRNA siti di legame. High-throughput sequenziamento del DNA CHIRP arricchito produce una mappa globale di lncRNA siti di legame. La Drosophila lncRNA roX2 è noto per interagire con il cromosoma X in un modo che è necessaria la compensazione del dosaggio. Figura 4 mostra roX2 profilo di legame su una sezione del cromosoma X. Sia &quot;anche&quot; e &quot;dispari&quot; i campioni sono stati sequenziati e le loro unici rumori sono stati eliminati per produrre una traccia di segnali sovrapposti. Ogni &quot;picco&quot; indica qui un sito di forte roX2 vincolante. La pista e la lista delle roX2 geni bersaglio sono stati descritti in Chu et al. 2011 17. <img alt="Figura 1." src="/files/ftp_upload/3912/3912fig1.jpg" /> Figura 1. Diagramma di flusso della procedura CHIRPdure. La cromatina è reticolata ad lncRNA: addotti proteici in vivo Biotinylated sonde piastrelle sono ibridate per indirizzare lncRNA e complessi della cromatina sono purificati utilizzando perline streptavidina magnetici, seguiti da lavaggi stringenti.. Noi eluire DNA legato lncRNA o proteine, con un cocktail di RNasi A e H. è schematizzata Una sequenza putativa lncRNA vincolante in arancione. Precedentemente pubblicato in Chu et al. 2011 17. <img alt="Figura 2." src="/files/ftp_upload/3912/3912fig2.jpg" /> Figura 2. Arricchisce CHIRP per l&#39;uomo TERC RNA. TERC-asDNA sonde recuperare ~ 88% dei cellulari TERC RNA non rilevabile e GAPDH. LacZ-asDNA sonde vengono utilizzati come controlli negativi e recuperare né RNA. Media + sd vengono visualizzati. Precedentemente pubblicato in Chu et al. 2011 17. <img alt="Figura 3." src="/files/ftp_upload/3912/3912fig3.jpg" /> Figura 3. Hotair CHIRP-qPCR in umana primaria perfibroblasti Eskin. NFKBIA, HOXD3-4, SERINC5 e ABCA2 sono regioni che interagiscono con Hotair. TERC e GAPDH serviti come controlli negativi. Media + sd vengono visualizzati. Precedentemente pubblicato in Chu et al. 2011 17. <img alt="Figura 4." src="/files/ftp_upload/3912/3912fig4.jpg" /> Figura 4. CHIRP-seguenti dati di roX2 SL2 RNA in cellule di Drosophila. &quot;Anche&quot; e &quot;dispari&quot; sono stati sequenziati separatamente, i loro dati si fondono in modo da riflettere solo picchi comuni in entrambi. La pista è mostrato unito. Precedentemente pubblicato in Chu et al. 2011 17.

<ol>
	<li>Koziol, M. J., Rinn, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+traffic+control+of+chromatin+complexes.">RNA traffic control of chromatin complexes.</a> Curr. Opin. Genet. Dev. 20, 142-148 (2010).</li><li>Mercer, T. R., Dinger, M. E., Mattick, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+non-coding+RNAs:+insights+into+functions.">Long non-coding RNAs: insights into functions.</a> Nat. Rev. Genet. 10, 155-159 (2009).</li><li>Rinn, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+demarcation+of+active+and+silent+chromatin+domains+in+human+HOX+loci+by+noncoding+RNAs.">Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs.</a> Cell. 129, 1311-1323 (2007).</li><li>Zhao, J., Sun, B. K., Erwin, J. A., Song, J. J., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycomb+proteins+targeted+by+a+short+repeat+RNA+to+the+mouse+X+chromosome.">Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome.</a> Science. 322, 750-756 (2008).</li><li>Kelley, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+spreading+of+the+Drosophila+dosage+compensation+complex+from+roX+RNA+genes+into+flanking+chromatin.">Epigenetic spreading of the Drosophila dosage compensation complex from roX RNA genes into flanking chromatin.</a> Cell. 98, 513-522 (1999).</li><li>Pandey, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kcnq1ot1+antisense+noncoding+RNA+mediates+lineage-specific+transcriptional+silencing+through+chromatin-level+regulation.">Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation.</a> Mol. Cell. 32, 232-246 (2008).</li><li>Wang, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+long+noncoding+RNA+maintains+active+chromatin+to+coordinate+homeotic+gene+expression.">A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression.</a> Nature. 472, 120-124 (2011).</li><li>Khalil, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Many+human+large+intergenic+noncoding+RNAs+associate+with+chromatin-modifying+complexes+and+affect+gene+expression.">Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 11667-11672 (2009).</li><li>Zhao, J. <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+identification+of+polycomb-associated+RNAs+by+RIP-seq.">Genome-wide identification of polycomb-associated RNAs by RIP-seq.</a> Mol. Cell. 40, 939-953 (2010).</li><li>Meller, V. H., Wu, K. H., Roman, G., Kuroda, M. I., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=roX1+RNA+paints+the+X+chromosome+of+male+Drosophila+and+is+regulated+by+the+dosage+compensation+system.">roX1 RNA paints the X chromosome of male Drosophila and is regulated by the dosage compensation system.</a> Cell. 88, 445-457 (1997).</li><li>Franke, A., Baker, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rox1+and+rox2+RNAs+are+essential+components+of+the+compensasome,+which+mediates+dosage+compensation+in+Drosophila.">The rox1 and rox2 RNAs are essential components of the compensasome, which mediates dosage compensation in Drosophila.</a> Mol. Cell. 4, 117-122 (1999).</li><li>Gupta, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+non-coding+RNA+HOTAIR+reprograms+chromatin+state+to+promote+cancer+metastasis.">Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis.</a> Nature. 464, 1071-1076 (2010).</li><li>Tsai, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+noncoding+RNA+as+modular+scaffold+of+histone+modification+complexes.">Long noncoding RNA as modular scaffold of histone modification complexes.</a> Science. 329, 689-693 (2010).</li><li>Zappulla, D. C., Cech, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+as+a+flexible+scaffold+for+proteins:+yeast+telomerase+and+beyond.">RNA as a flexible scaffold for proteins: yeast telomerase and beyond.</a> Cold Spring Harb. Symp. Quant. Biol.. 71, 217-224 (2006).</li><li>Nagano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Air+noncoding+RNA+epigenetically+silences+transcription+by+targeting+G9a+to+chromatin.">The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin.</a> Science. 322, 1717-1720 (2008).</li><li>Carter, D., Chakalova, L., Osborne, C. S., Dai, Y. F., Fraser, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-range+chromatin+regulatory+interactions+in+vivo.">Long-range chromatin regulatory interactions in vivo.</a> Nature. 32, 623-626 (2002).</li><li>Chu, C., Qu, K., Zhong, F. L., Artandi, S. E., Chang, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+Maps+of+Long+Noncoding+RNA+Occupancy+Reveal+Principles+of+RNA-Chromatin+Interactions.">Genomic Maps of Long Noncoding RNA Occupancy Reveal Principles of RNA-Chromatin Interactions.</a> Mol. Cell. , (2011).</li><li>Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+individual+mRNA+molecules+using+multiple+singly+labeled+probes.">Imaging individual mRNA molecules using multiple singly labeled probes.</a> Nat. Methods. 5, 877-879 (2008).</li></ol>

C. Chu e HY Chang sono denominati come inventori su una domanda di brevetto basato su questo metodo.

Qui abbiamo descritto CHIRP-seq, un metodo di mappatura in vivo i siti di legame lncRNA genome-wide. I parametri chiave per il successo sono le piscine separate di piastrelle sonde oligonucleotidiche e reticolazione glutaraldeide. Il disegno di sonde per affinità è semplice data la sequenza di RNA e non richiede alcuna conoscenza preventiva della struttura del RNA o domini funzionali. Il nostro successo con roX2, TERC e Hotair - tre RNA piuttosto differenti in due specie - suggerisce che CHIRP-seq è probabile generalizzabile a molte lncRNAs. Come per tutti gli esperimenti, i controlli per la cura e propria sono tenuti a interpretare i risultati. LncRNA diversi possono richiedere la titolazione delle condizioni, giudizioso e il cambiamento delle condizioni, come la selezione di sonde affinità diverse o reticolanti, può mettere in luce aspetti diversi di interazioni RNA-cromatina. Come ChIP-seq, non tutti gli eventi vincolanti sono necessariamente funzionali, e sono necessari ulteriori studi per accertare le conseguenze biologiche di RNA occupancy sulla cromatina. Tuttavia, si prevede molte interessanti applicazioni di questa tecnologia per i ricercatori di altri cromatina associate lncRNAs, che ora si contano a migliaia 8,9. Proprio come ChIP-seq ha aperto la porta per la genome-wide esplorazioni di interazioni proteina-DNA, CHIRP-seq studi della &quot;RNA interattoma&quot; può rivelare molte nuove vie della biologia.

Buffer List:
Dissolve a pellet of complete protease inhibitor in 1 ml water as 50x stock. Make 100 mM PMSF in isopropanol (100x stock). Superase-in is used as 200x stock. Store all at -20&deg;C.
Lysis Buffer:
50 mM Tris-Cl pH 7.0 
10 mM EDTA 
1% SDS 
Always add PMSF, P.I. and Superase-in fresh before use except when washing beads
Proteinase K Buffer (for DNA)
100 mM NaCl 
10 mM TrisCl pH 8.0 (For RNA use pH 7.0) 
1 mM EDTA 
0.5% SDS 
Add 5% by volume Proteainse K (Ambion AM2546 20 mg/ml) fresh before use
Hybridization Buffer
750 mM NaCl 
1% SDS 
50 mM Tris-Cl pH 7.0 
1 mM EDTA 
15% formamide (store in the dark at 4&deg;C) 
Always add PMSF, P.I. and Superase-in fresh before use
Wash Buffer
2x NaCl and Sodium citrate (SSC) (diluted from 20x SSC Invitrogen stock) 
0.5% SDS 
Always add PMSF fresh before use
DNA elution Buffer
50 mM NaHCO3 
1% SDS
Table of specific reagents and equipment:
<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Glutaraldehyde (EM grade)</td>
			<td>Sigma Aldrich</td>
			<td>G5882-10x10ml</td>
		</tr>
		<tr>
			<td>Motorized pellet mixer</td>
			<td>VWR</td>
			<td>V8185-904</td>
		</tr>
		<tr>
			<td>Protease inhibitor</td>
			<td>Roche</td>
			<td>11873580001</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma Aldrich</td>
			<td>78830</td>
		</tr>
		<tr>
			<td>Superase-in</td>
			<td>Ambion</td>
			<td><a href="http://www.ambion.com/catalog/CatNum.php?AM2696">AM2696</a></td>
		</tr>
		<tr>
			<td>Bioruptor</td>
			<td>Diagenode</td>
			<td>UCD-200</td>
		</tr>
		<tr>
			<td>Falcon tubes (for sonication)</td>
			<td>Corning</td>
			<td>430790</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Ambion</td>
			<td>AM2546</td>
		</tr>
		<tr>
			<td>PCR purification kit</td>
			<td>Qiagen</td>
			<td>28106</td>
		</tr>
		<tr>
			<td>C-1 magnetic beads</td>
			<td>Invitrogen</td>
			<td>65002</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma Aldrich</td>
			<td>P7626-25G</td>
		</tr>
		<tr>
			<td>DynaMag-15 magnet</td>
			<td>Invitrogen</td>
			<td>123-01D</td>
		</tr>
		<tr>
			<td>DynaMag-2 magnet</td>
			<td>Invitrogen</td>
			<td>123-21D</td>
		</tr>
		<tr>
			<td>MIRNeasy mini kit</td>
			<td>Qiagen</td>
			<td>217004</td>
		</tr>
		<tr>
			<td>Rnase H</td>
			<td>Epicentre</td>
			<td>R0601K</td>
		</tr>
		<tr>
			<td>Rnase A</td>
			<td>Sigma Aldrich</td>
			<td>R4875-100MG</td>
		</tr>
		<tr>
			<td>Phase Lock Gel Heavy</td>
			<td>5 PRIME</td>
			<td>2302810</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Invitrogen</td>
			<td>15596-018</td>
		</tr>
		<tr>
			<td>Phenol:chloroform:Isoamyl</td>
			<td>Invitrogen</td>
			<td>15593-031</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Ricca</td>
			<td>RSOC0020-1C</td>
		</tr>
		<tr>
			<td>GlycoBlue</td>
			<td>Ambion</td>
			<td>AM9515</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>J.T. Baker</td>
			<td>4057-06</td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Invitrogen</td>
			<td>10010-049</td>
		</tr>
		<tr>
			<td>Elution Buffer (EB)</td>
			<td>Qiagen</td>
			<td>19086</td>
		</tr>
		<tr>
			<td>20x SSC</td>
			<td>Invitrogen</td>
			<td>15557-036</td>
		</tr>
		<tr>
			<td>10% SDS</td>
			<td>Invitrogen</td>
			<td>15553-027</td>
		</tr>
		<tr>
			<td>DNA-free</td>
			<td>Ambion</td>
			<td>AM1906</td>
		</tr>
		<tr>
			<td>Buffer kit</td>
			<td>Ambion</td>
			<td>AM9010</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Invitrogen</td>
			<td>15515-026</td>
		</tr>
	</tbody>
</table>

chromatin isolation by rna purification (chirp)

RNA non codificanti lunghi sono regolatori chiave degli Stati cromatina per importanti processi biologici quali la compensazione del dosaggio, imprinting, e l&#39;espressione genica di sviluppo 1,2,3,4,5,6,7. La recente scoperta di migliaia di lncRNAs in associazione con specifici complessi di modifica della cromatina, come Polycomb Complex repressiva 2 (PRC2) che media l&#39;istone H3 lisina 27 trimethylation (H3K27me3), suggerisce i ruoli di massima per la gestione lncRNAs numerosi stati della cromatina in un gene specifico moda 8,9. Mentre alcuni lncRNAs sono pensati per lavorare in cis sui geni vicini, lncRNAs altri lavorano in trans per regolare i geni si trovano lontano. Per esempio, Drosophila lncRNAs roX1 e roX2 legano numerose regioni del cromosoma X delle cellule maschili, e sono fondamentali per la compensazione del dosaggio 10,11. Tuttavia, le posizioni esatte dei loro siti di legame non sono note ad alta risoluzione. Allo stesso modo, umano lncRNA Hotair può influire sulla occupazione PRC2 hundreds di geni genoma 3,12,13, ma come specificità si ottiene non è chiaro. LncRNAs può anche servire come strutture modulari di assumere l&#39;assemblaggio di complessi proteici multipli. Il classico trans-agente RNA scaffold è il TERC RNA che serve come modello e scaffold per la telomerasi complesso 14; Hotair può anche servire come impalcatura per PRC2 e H3K4 demetilasi complesso 13. Precedenti studi di mappatura occupazione RNA a cromatina hanno rivelato notevoli intuizioni 15,16, ma solo in un locus singolo gene alla volta. I siti di occupazione della maggior parte dei lncRNAs non sono noti, ei ruoli dei lncRNAs nella regolazione della cromatina sono stati in gran parte dedotta dagli effetti indiretti della perturbazione lncRNA. Proprio come immunoprecipitazione della cromatina seguita da microarray o sequenziamento profondo (ChIP-chip o ChIP-seq, rispettivamente) ha notevolmente migliorato la nostra comprensione di interazioni proteina-DNA su scala genomica, qui illustrare un RecenTLY pubblicato strategia per mappare lunga occupazione RNA genome-wide ad alta risoluzione 17. Questo, Isolamento metodo cromatina di purificazione dell&#39;RNA (CHIRP) (Figura 1), si basa sulla cattura affinità di bersaglio lncRNA: complesso cromatina allineando-oligo antisenso, che genera un mappa genomica di siti di legame ad una risoluzione di diverse centinaia di basi con alta sensibilità e basso background. CHIRP è applicabile a molti lncRNAs perché il progetto di affinità sonde è semplice data la sequenza di RNA e non richiede alcuna conoscenza della struttura del RNA o domini funzionali.

Watch this Scientific Journal Video about Chromatin Isolation by RNA Purification ChIRP at JoVE.com

Chromatin Isolation by RNA Purification ChIRP

CHIRP è una tecnica innovativa e rapida mappa genomica siti di legame di RNA non codificanti lunghi (lncRNAs). Il metodo sfrutta la specificità di oligonucleotidi anti-senso di affiancamento per consentire l&#39;enumerazione di lncRNA-bound siti genomici.

Isolamento di cromatina Purificazione RNA (disturbo)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

chromatin-isolation-by-rna-purification-chirp

Research

JoVE Journal

Biology

Chromatin Isolation by RNA Purification (ChIRP)

85.7K Views.  Stanford University School of Medicine. CHIRP è una tecnica innovativa e rapida mappa genomica siti di legame di RNA non codificanti lunghi (lncRNAs). Il metodo sfrutta la specificità di oligonucleotidi anti-senso di affiancamento per consentire l'enumerazione di lncRNA-bound siti genomici.

Video: Chromatin Isolation by RNA Purification ChIRP

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

La colorazione delle proteine ​​in gel con Coomassie G-250, senza solventi organici acido acetico e

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Ricerca

Biologia

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Isolamento e purificazione di Drosophila Neuroni periferici da Magnetic Ordinamento Bead

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

Generazione di DNA / RNA ibrido nel DNA genomico da trasformazione utilizzando RNA contenenti oligonucleotidi

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

Isolamento e caratterizzazione di RNA contenenti esosomi

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

In Endocitosi Fegato vivo seguita da purificazione delle cellule del fegato da parte di perfusione epatica

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Isolamento e purificazione di chinesina dal Drosophila Embrioni

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

Trascrizione in vitro e tappatura mRNA Luciferase Gaussia Seguito da cellule HeLa Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Isolamento rapida e purificazione di mitocondri per il trapianto da tessuto Dissociazione e Filtrazione Differenziale

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

Generazione e Purificazione di umani INO80 rimodellamento della cromatina Complessi e Sottocomplessi

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Isolamento dell&#39;RNA da mouse Pancreas: Un Tessuto ricco Ribonucleasi

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...