Questo studio è stato sostenuto da sovvenzioni da Taiwan (Ministero della scienza e della tecnologia, più 100-2923-B-002-002-MY3 e 106-2313-B-002-011-MY3 a H.J.L.), Repubblica Ceca (concedere Università Agenzia della Boemia meridionale, GAJU concedere 065/2017/P a Y.H.L) e Spagna ( Ministero spagnolo dell'economia e della competitività, concede CGL2012-36251 e CGL2015-64727-P a X.B. e il governo catalano, concedere 2014 SGR 619 a X.B.); ha inoltre ricevuto il sostegno finanziario del Fondo europeo economica e lo sviluppo regionale (fondi FEDER a X.B.).

1. sintesi e preparazione di dsRNA
<ol><li>Identificare i siti di destinazione di dsRNA nella regione 3' non tradotta dei geni bersaglio. La dsTub è utilizzato per il targeting il gene α-tubulina (tub) (numero di accessione GenBank: KX228233), e dsEGFP come un controllo negativo dsRNA è progettato dalla sequenza di intensificare la proteina fluorescenza verde (EGFP; Numero di accessione GenBank: LC311024).</li>
	<li>Eseguire l'amplificazione di PCR standard per sintetizzare i modelli di dsRNA con gli iniettori di gene-specific contenente la sequenza del promotore T7 (5'-TAATACGACTCACTATAGGG-3'). Le condizioni di amplificazione di PCR e informazioni sul primer utilizzati sono quelli segnalati da Lin et al. (2017) 12 (Vedi Tabella materiali).</li>
	<li>Procedere con la preparazione di dsRNA utilizzando una trascrizione di T7 Kit (Vedi Tabella materiali), seguendo le istruzioni del produttore. Nota: Resa elevata quantità dsRNA, mescolare due reazioni in un tubo (in totale 40 µ l) e incubare a 37 ° C durante la notte.</li>
	<li>Aggiungere 2 µ l di dnasi (Vedi Tabella materiali) in campioni di dsRNA per 15 min a 37 ° C per modelli del DNA del digest.</li>
	<li>Aggiungere 200 µ l di reagente di estrazione (Vedi Tabella materiali) e cloroformio 40 µ l, poi vortice.</li>
	<li>Centrifugare per 10 min a 13.000 x g a 4 ° C, quindi raccogliere il surnatante (150 µ l) in un nuovo tubo del microcentrifuge.</li>
	<li>Precipitare dsRNA aggiungendo 150 µ l isopropanolo e incubare per 15 minuti in ghiaccio.</li>
	<li>Centrifugare per 15 min a 13.000 x g a 4 ° C, quindi rimuovere il surnatante.</li>
	<li>Aggiungere 200 µ l di etanolo 70% per lavare dsRNA pellet. Centrifugare per 10 min a 13.000 x g a 4 ° C.</li>
	<li>Rimuovere l'etanolo e ripetere le fasi di lavaggio di etanolo.</li>
	<li>Rimuovere l'etanolo e asciutto dsRNA pellet da concentratori centrifughi vuoto per 3 min, quindi risospendere dsRNA con acqua RNAsi-libera.</li>
	<li>Determinare la quantità del dsRNA purificato utilizzando uno spettrofotometro UV-Vis di micro-volume (Vedi Tabella materiali) secondo le istruzioni del produttore e regolare fino alla concentrazione desiderata (ad es., 0,25 µ g / µ l per la preparazione di dsRNA lipoplessi).</li>
	<li>Conservare i campioni di dsRNA a-20 ° C fino a 3 mesi.</li>
</ol>2. preparazione del dsRNA lipoplessi
<ol><li>Diluire 4 µ l di reagente di transfezione (Vedi Tabella materiali) aggiungendo 4 µ l di soluzione glucosata al 5%. Diluire 4 µ l di dsRNA (1 µ g) aggiungendo 4 µ l di soluzione glucosata al 5%.</li>
	<li>Aggiungere la soluzione di reagente liposoma diluito immediatamente nella soluzione diluita dsRNA tutti in una volta. Vortice brevemente per mescolarli, quindi incubare per 15 minuti a 25 ° C. Nota: Non mescolare le soluzioni nell'ordine inverso.</li>
	<li>I dsRNA lipoplessi sono pronti all'uso. Usare entro 1 ora di preparazione e scartare dopo 1 ora. Nota: Secondo le istruzioni del produttore, il lipoplessi dovrebbero essere usato appena possibile.</li>
</ol>3. raccolta di enzimi extracellulari dall'emolinfa e succo del Midgut
<ol><li>
Insetto buffer saline (stock x 10)
	<ol>
<li>Mescolare 900 mL acqua distillata doppia con 109,3 g NaCl, 15,7 g KCl, 6,3 g CaCl2e 8,3 g MgCl2·6H2O.</li>
		<li>Aggiustare il pH a 6,8 e riempire fino a 1.000 mL.</li>
	</ol>
</li>
	<li>
Collezione di emolinfa
	<ol>
<li>Anestetizzare gli scarafaggi sul ghiaccio fino a quando non si muovono per 3 min.</li>
		<li>Tenere lo scarafaggio con due dita (pollice e indice) e alzare il lato ventrale del scarafaggio.</li>
		<li>Utilizzare le forbici per dissezione per tagliare fuori la punta di una coxa di una gamba posteriore. Nota: Tagliare l'intersezione collegato tra la coxa e il trocantere. L'asportazione della parte del coxa era meno efficiente per raccogliere l'emolinfa.</li>
		<li>Spremere l'addome delicatamente con il dito medio e raccogliere l'emolinfa sanguinamento dall'incisione con una micropipetta 10 µ l.</li>
		<li>Piscina l'emolinfa da scarafaggi (5 persone) in un tubo del microcentrifuge. Nota: Mantenere le provette microcentrifuga sul ghiaccio per prevenire melanizzazione. Il volume medio di emolinfa ottenuto da uno scarafaggio maschio è 2-3 µ l.</li>
		<li>Rotazione verso il basso l'emolinfa brevemente con una centrifuga da banco per 10 s.</li>
		<li>Trasferire il volume desiderato di emolinfa (vale a dire, 10 µ l) e mescolare con 50 µ l di tampone salino insetto 1x.</li>
		<li>Centrifugare per 10 minuti a 1.000 x g a 4 ° C a girare giù emociti. Trasferire il surnatante in una provetta pulita microcentrifuga.</li>
		<li>Quantificare la concentrazione di proteine totali utilizzando uno spettrofotometro UV-Vis di micro-volume misurando A28015.</li>
		<li>Regolare ogni campione per avere la stessa concentrazione di proteina (6 mg / µ l di proteina totale).</li>
	</ol>
</li>
	<li>
Raccolta di succo del midgut
	<ol>
<li>Anestetizzare gli scarafaggi sul ghiaccio. Sistemare gli scarafaggi lato ventrale sulla piastra di dissezione con freddo 1x insetto Salina buffer mediante spilli entomologici.</li>
		<li>Sezionare l'addome con una pinzetta bene. Rimuovere l'intestino intero (compreso l'intestino anteriore, media e posteriore) per un piatto fresco con 1 x insetto tampone salino.</li>
		<li>Rimuovere l'intestino anteriore e posteriore e di trasferire velocemente midgut ad un tubo del microcentrifuge che contiene 100 µ l di tampone salino dell'insetto.</li>
		<li>Piscina l'intestino da sei scarafaggi nel tubo stesso e vortexare per 10 s.</li>
		<li>Centrifugare per 10 minuti a 1.000 x g a 4 ° C per rallentare gli emociti e tessuti di intestino. Trasferire il surnatante in una provetta pulita microcentrifuga.</li>
		<li>Quantificare la concentrazione di proteine totali utilizzando uno spettrofotometro UV-Vis di micro-volume misurando A28015.</li>
		<li>Regolare ogni campione per avere la stessa concentrazione di proteina (6 mg / µ l di proteina totale).</li>
	</ol>
</li>
</ol>4. dsRNA degradazione Assay
<ol><li>Appena preparare 4 µ l del dsRNA nudo o lipoplessi dsRNA (1 µ g).</li>
	<li>Mescolare il succo di dsRNA enzimi di soluzioni con 10 µ l di tampone salino dell'insetto (controllo), estratti dall'emolinfa, o del midgut.</li>
	<li>Aggiungere 2 µ l di EGTA (20 mM) o acqua RNAsi-libera per separare campioni come controlli per inibizione delle attività enzimatiche.</li>
	<li>Incubare per 1 ora o più a lungo (6, 12 o 24 ore) a 25 ° C.</li>
	<li>Aggiungere 200 µ l di reagente di estrazione e cloroformio 40 µ l, poi vortice.</li>
	<li>Centrifugare per 10 min a 13.000 x g a 4 ° C, quindi raccogliere il surnatante (150 µ l) in un nuovo tubo del microcentrifuge.</li>
	<li>Precipitare dsRNA aggiungendo 150 µ l isopropanolo e incubare per 15 minuti in ghiaccio.</li>
	<li>Centrifugare per 15 min a 13.000 x g a 4 ° C, quindi rimuovere il surnatante.</li>
	<li>Aggiungere 200 µ l di etanolo 70% per lavare dsRNA pellet. Centrifugare per 10 min a 13.000 x g a 4 ° C.</li>
	<li>Rimuovere l'etanolo e ripetere le fasi di lavaggio di etanolo.</li>
	<li>Rimuovere l'etanolo e asciutto dsRNA pellet da concentratori centrifughi vuoto per 3 min. Risospendere dsRNA con acqua RNAsi-libera di 10 µ l.</li>
	<li>Verificare l'integrità del dsRNA Trattato tramite elettroforesi su gel di agarosio 1.5%.</li>
</ol>5. la somministrazione orale di dsRNA
<ol><li>Mantenere gli scarafaggi su una dieta ad libitum di cibo secco (chow cane). Privare l'approvvigionamento idrico dagli scarafaggi un giorno prima gli esperimenti di ingestione di dsRNA.</li>
	<li>Tenere uno scarafaggio afferrando le ali con le pinze flessibili (Figura 1, punto 5). Nota: Non afferrare le parti del corpo di scarafaggi.</li>
	<li>Preparare il dsRNA lipoplessi, come pure di dsRNA nuda, per l'alimentazione, come descritto nella sezione 2. Nota: Il dsRNA lipoplessi devono essere preparata fresca prima della poppata.</li>
	<li>Alimentazione 4 µ l di dsRNA lipoplessi o nudo dsRNA (250 ng) con una micropipetta.</li>
	<li>Lentamente dispensare la goccia della soluzione dsRNA vicino all'apparato boccale di scarafaggi e lasciare che gli scarafaggi ingerire la goccia completamente.</li>
	<li>Eseguire gli esperimenti di ingestione due volte al giorno (1 h dopo luci su e 1 h prima luci spente) per 8 giorni o 16 giorni di continuo. Nota: Tutte le blatte acquisito acqua solo tramite alimentazione manuale con reagenti soluzione o controllo di dsRNA (ad es., acqua gratuita nucleasi, soluzione di glucosio e reagente liposoma) durante gli esperimenti di ingestione.</li>
	<li>Fornire bottiglie d'acqua per gli scarafaggi dopo 16 giorni di ingestione di dsRNA esperimenti.</li>
</ol>6. valutare atterramento
<ol><li>Al momento scelto punti (2, 9 e 17 giorni dopo il test di ingestione di dsRNA), raccogliere gli insetti dsEGFP - e dsTub-trattati e sezionare i tessuti selezionati (per esempio, del midgut).</li>
	<li>Eseguire PCR quantitativa in tempo reale (qRT-PCR). Le condizioni di amplificazione qRT-PCR e informazioni sul primer come riportato da Lin et al. (2017) 12.</li>
	<li>Valutare il controllo e il dsRNA-Trattato insetti ogni giorno per controllare la mortalità e rimuovere gli scarafaggi che sono più in movimento.</li>
</ol>

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Questo protocollo presenta un metodo per RNAi efficace attraverso la consegna orale di dsRNA lipoplessi, che comprende la protezione contro la digestione di ribonucleasi nel succo del midgut il tedesco scarafaggio. Come dimostrato in altri studi in varie specie di insetti, l'effetto di RNAi povero attraverso la consegna orale di dsRNA è rappresentato principalmente dalla degradazione di dsRNA8,9,10. Questo protocollo produce li...

RNAi è stato dimostrato come un metodo efficace per l'espressione genica atterramento attraverso un meccanismo di silenziamento post-trascrizionale via innescato da molecole di dsRNA in molti eucarioti1. Nell'ultimo decennio di studi, RNAi è diventata uno strumento utile per studiare le funzioni dei geni dallo sviluppo di comportamento che riducono l'espressione di specifici geni tramite iniezione e/o alimentazione di dsRNA in vari taxa di insetti2,3. A causa della specificità e robustezza dell'effetto di svuotamento, l'applicazione di RNAi è attualmente considerato come una strategia potenziale per pest control gestione4,5. Tuttavia, l'efficienza di RNAi varia ampiamente tra specie di insetti, a seconda di diversi geni presi di mira e i metodi di consegna. Un corpo crescente di prova suggerisce che l'instabilità del dsRNA, che è stato degradato dalla ribonucleasi, è un fattore critico per la limitata efficacia della RNAi5,6. Per esempio, la bassa sensibilità di RNAi in Manduca sexta è stata spiegata dal fatto che il dsRNA mescolato con l'emolinfa è stato rapidamente degradato entro 1 ora7. Allo stesso modo, la presenza di nucleasi alcaline in midgut, che degradano in modo efficiente dsRNA ingerito, è fortemente correlata con bassa efficienza di RNAi in diversi ordini di insetti8,9,10.
La somministrazione orale di dsRNA è particolarmente interessante per l'applicazione del RNAi in una strategia di controllo dei parassiti, ma un metodo per ritardare la degradazione di dsRNA di nucleasi a midgut non è ancora stato sviluppato, che avrebbe il potenziale per garantire un efficace RNAi attraverso l'alimentazione. Tuttavia, il problema del blocco di RNAi per consegna orale di dsRNA è stato segnalato dalla grande quantità di dsRNA, ad es. 50 µ g in Bombyx mori, o alimentazione alimentazione continuamente per 8 giorni (8 µ g dsRNA in totale) nelle specie di locusta. Il tedesco scarafaggio, germinica di Blattella, è altamente sensibile alla RNAi tramite l'iniezione del dsRNA11,12,13,14, ma non è rispondente a dsRNA attraverso l'alimentazione. Recentemente, Lin et al. (2017) hanno dimostrato che il dsRNA incapsulato con risultati di vettori liposomi in successo RNAi per atterramento l'espressione genica α-tubulina nella mortalità significativa del midgut e trigger del tedesco scarafaggio15. Come la degradazione di dsRNA in midgut è il fattore limitante per RNAi orale, i vettori di liposomi servono come veicolo di proteggere dsRNA dalla degradazione, che è facilmente applicabile in altri insetti con le attività della nucleasi forte nell'intestino. Della nota, la ragione per la scelta del reagente di transfezione particolare (Vedi Tabella materiali) abbiamo usato come vettore di liposomi nel protocollo attuale è che è stato testato per la transfezione di cellule di insetto linea con meno tossicità, secondo la istruzioni del produttore. Secondo il confronto dei sistemi di transfezione diversi liposoma Gharavi et al. (2013) 16, l'efficienza di trasfezione Short interfering RNA (siRNA) è circa la stessa tra questo e altri sistemi disponibili in commercio che sono stati utilizzati per sistemi di dsRNA in altri insetti17,18 . Inoltre, il nostro metodo di alimentazione sia abbastanza attento a garantire la corretta quantità di dsRNA è ingerita da ogni scarafaggio, e che i risultati siano robusti e confermato. In sintesi, il presente protocollo e i risultati dimostrano che utilizzando dsRNA lipoplessi migliora la stabilità di dsRNA e apre la porta alla progettazione della consegna orale strategia di RNAi, che è un approccio promettente per controllo dei parassiti in futuro.

<table border="0" cellpadding="0" cellspacing="0" style="width:1124px;" width="1123">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">GenJe Plus DNA in vitro Transfection reagent</td>
			<td style="width:133px;">SignaGen</td>
			<td style="width:157px;">SL100499</td>
			<td style="width:481px;">for lipoplexes preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blend Taq plus</td>
			<td style="width:133px;">TOYOBO&#160;</td>
			<td style="width:157px;">BTQ-201</td>
			<td>for PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast SYBR Green Master Mix</td>
			<td style="width:133px;">ABI&#160;</td>
			<td style="width:157px;">4385612</td>
			<td>for qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">FirstChoice RLM-RACE Kit</td>
			<td style="width:133px;">Invitrogen</td>
			<td style="width:157px;">AM1700</td>
			<td>for 3&#39; UTR identification&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">MEGAscript T7 Transcription Kit</td>
			<td style="width:133px;">Invitrogen</td>
			<td style="width:157px;">AMB13345</td>
			<td>for dsRNA synthesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TURBO DNase</td>
			<td style="width:133px;">Invitrogen</td>
			<td style="width:157px;">AM2239</td>
			<td>remove DNA template from dsRNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRIzol</td>
			<td style="width:133px;">Invitrogen</td>
			<td style="width:157px;">15596018</td>
			<td>for dsRNA or total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RQ1 RNase-Free Dnase</td>
			<td style="width:133px;">Promega</td>
			<td style="width:157px;">M6101</td>
			<td>remove DNA template from total RNA&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">chloroform&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">C2432</td>
			<td>for dsRNA or total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">2-Propanol</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">I9516</td>
			<td>for dsRNA or total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>24102</td>
			<td>for dsRNA or total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Diethyl pyrocarbonate, DEPC</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">D5758</td>
			<td>for RNase free water preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">glucose solution</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">G3285</td>
			<td>for lipoplexes preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride, NaCl</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">S7653</td>
			<td>insect saline buffer formula</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Potassium chloride, KCl</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">P9333</td>
			<td>insect saline buffer formula</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:352px;">Calcium chloride, CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
			<td>insect saline buffer formula</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Magnesium chloride hexahydrate, MgCl2.6H2O</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">M2670</td>
			<td>insect saline buffer formula</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">EGTA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">E3889</td>
			<td>enzyme inhibitor&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dissecting scissor</td>
			<td style="width:133px;">F.S.T.</td>
			<td style="width:157px;"></td>
			<td>cockroach dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fine tweezers</td>
			<td style="width:133px;">F.S.T.</td>
			<td style="width:157px;"></td>
			<td>cockroach dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">flexible tweezer</td>
			<td style="width:133px;">F.S.T.</td>
			<td style="width:157px;"></td>
			<td>cockroach holding&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pipetman</td>
			<td style="width:133px;">RAININ</td>
			<td style="width:157px;">P10</td>
			<td>sample preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microcentrifuge tube</td>
			<td style="width:133px;">Axygen</td>
			<td>MCT175C, PCR02C</td>
			<td>sample preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">pipette tip&#160;</td>
			<td style="width:133px;">Axygen</td>
			<td></td>
			<td>sample preparation</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">vortexter</td>
			<td style="width:133px;">Digisystem</td>
			<td style="width:157px;">vm1000</td>
			<td>sample preparation</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Minispin centrifuge</td>
			<td style="width:133px;">The Gruffin Group</td>
			<td style="width:157px;">GMC 206</td>
			<td>for liquid spin down&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td style="width:133px;">ALC</td>
			<td style="width:157px;">PK121R</td>
			<td>sample preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pH meter&#160;</td>
			<td style="width:133px;">JENCO</td>
			<td style="width:157px;">6071</td>
			<td>for pH adjust</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">micro-volume spectrophotometer</td>
			<td style="width:133px;">Quawell</td>
			<td style="width:157px;">Q3000</td>
			<td>nucleic acid quantitative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">PCR Thermal cycler</td>
			<td style="width:133px;">ABI&#160;</td>
			<td style="width:157px;">2720</td>
			<td>for template PCR or&#160; dsRNA synthesis incubation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">quantitative real-time PCR</td>
			<td style="width:133px;">ABI&#160;</td>
			<td style="width:157px;">StepOne plus</td>
			<td>gene expression quantitative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifugal Vacuum Concentrators</td>
			<td style="width:133px;">eppendorf</td>
			<td style="width:157px;">5301</td>
			<td>for dsRNA or total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multipette&#160;</td>
			<td style="width:133px;">eppendorf</td>
			<td style="width:157px;">xstream</td>
			<td>for real-time PCR sample loading&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose I</td>
			<td style="width:133px;">amresco</td>
			<td style="width:157px;">0710</td>
			<td>for nucleic acid electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">tub gene specfifc forward preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>GGG ACA AGC CGG AGT GCA GA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">tub gene specfifc reverse preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>TCC TGC TCC TGT CTC GCT GA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">dsTub template forward primer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>TAA TAC GAC TCA CTA TAG GGA CAA GCC GGA GTG CAG&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">dsTub template reverse primer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>TAA TAC GAC TCA CTA TAG GGT CCT GCT CCT GTC TCG CTG&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">dsEGFP template forward preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>TAA TAC GAC TCA CTA TAG GGT ATG GTG AGC AAG GGC GAG GAG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">dsEGFP template reverse preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>TAA TAC GAC TCA CTA TAG GGT GGC GGA TCT TGA AGT TCA CC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">tub qPCR forward primer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>GGA CCG CAT CAG GAA ACT GGC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">tub qPCR reverse preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>CCA CAG ACA GCC TCT CCA TGA GC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">ef1 qPCR forward primer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>CGC TTG AGG AAA TCA AGA AGG A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">ef1 qPCRreverse preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>CCT GCA GAG GAA GAC GAA G</td>
		</tr>
	</tbody>
</table>

practical use of rna interference: oral delivery of double-stranded rna in liposome carriers for cockroaches

Interferenza del RNA (RNAi) è stato ampiamente applicato per scoprire le funzioni biologiche di numerosi geni ed è stata prevista come un parassita controllo strumento operativo tramite rottura di espressione genica essenziale. Sebbene diversi metodi, come l'iniezione, alimentazione e ammollo, sono stati segnalati per il recapito corretto del RNA double-stranded (dsRNA), l'efficienza di RNAi attraverso la consegna orale di dsRNA è altamente variabile tra i diversi gruppi degli insetti. Il tedesco scarafaggio, Blattella germanica, è altamente sensibile all'iniezione del dsRNA, come dimostrato da numerosi studi pubblicati in precedenza. Il presente studio descrive un metodo per dimostrare che il dsRNA incapsulato con vettori liposoma è sufficiente per ritardare la degradazione di dsRNA dal succo del midgut. In particolare, l'alimentazione in continuo di dsRNA incapsulato da liposomi significativamente riduce l'espressione di tubulina in midgut e ha portato alla morte di scarafaggi. In conclusione, la formulazione e l'utilizzo di dsRNA lipoplessi, che proteggono il dsRNA contro nucleasi, potrebbe essere in futuro un uso pratico di RNAi per controllo dei parassiti dell'insetto.

Uno schema semplificato del protocollo per la somministrazione orale di dsRNA è presentato nella Figura 1, dove sono mostrati i passaggi chiave per la preparazione di dsRNA lipoplessi.
Al fine di indagare la protezione fornita dai vettori liposomi al degrado di dsRNA nel succo del midgut di b. germanica, un'analisi ex vivo in cui si svolse la dsTub lipoplessi sono state incubate c...

Watch this Scientific Journal Video about Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches at JoVE.com

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

Uso pratico di interferenza del RNA: consegna orale del RNA Double-stranded in elementi portanti di liposomi per scarafaggi

Questo manoscritto viene illustrato lo svuotamento dell'espressione genica in midgut il tedesco scarafaggio attraverso ingestione orale di RNA double-stranded incapsulato in liposomi.

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

practical-use-rna-interference-oral-delivery-double-stranded-rna

Research

JoVE Journal

Biology

9.3K Views.  National Taiwan University. Questo manoscritto viene illustrato lo svuotamento dell'espressione genica in midgut il tedesco scarafaggio attraverso ingestione orale di RNA double-stranded incapsulato in liposomi.

Video: Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

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 ...

Ricerca

Biologia

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

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 ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

Generazione Substrato per endonucleasi di CRISPR / Cas Sistemi

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

Utilizzo di RNA-mediata strategia di alimentazione di interferenza secondo schermo per geni coinvolti nella regolazione Dimensione del corpo nel nematode<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

L&#39;esaurimento delle RNA ribosomiale per metagenomiche Gut Mosquito RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been made in identifying the components of the editosome complex that catalyze RNA editing. However, it is still not clear how those proteins work together. Chemical compounds obtained from a high-throughput screen against the editosome may block or affect one or more steps in the editing cycle. Therefore, the identification of new chemical compounds will generate valuable molecular probes for dissecting the editosome function and assembly. In previous studies, in vitro editing assays were carried out using radio-labeled RNA. These assays are time consuming, inefficient and unsuitable for high-throughput purposes. Here, a homogenous fluorescence-based &#8220;mix and measure&#8221; hammerhead ribozyme in vitro reporter assay to monitor RNA editing, is presented. Only as a consequence of RNA editing of the hammerhead ribozyme a fluorescence resonance energy transfer (FRET) oligoribonucleotide substrate undergoes cleavage. This in turn results in separation of the fluorophore from the quencher thereby producing a signal. In contrast, when the editosome function is inhibited, the fluorescence signal will be quenched. This is a highly sensitive and simple assay that should be generally applicable to monitor in vitro RNA editing or high throughput screening of chemicals that can inhibit the editosome function.

A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

RNA Catalyst come reporter per lo screening dei farmaci contro l&#39;RNA Editing in tripanosomi

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been ...

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle&#8217;s body cavity.
In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

RNA interference (RNAi)-based gene knockdown techniques are at the core of Tribolium research. Here, we provide an overview of our larval RNAi technique in Tribolium castaneum. Larval RNAi is a simple, but powerful technique that provides quick access to loss-of-function phenotypes, allowing researchers to study gene functions in diverse contexts.

Larvali interferenza RNA nella Red Farina Beetle,<em&gt; Tribolium castaneum</em

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully ...

Production of Double-stranded DNA Ministrings

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ministrings possess a heightened safety profile and also efficiently deliver DNA cargo to targeted cells. Conventional DNA vectors carry undesirable prokaryotic sequences, including antibiotic resistance genes, CpG motifs, and bacterial origins of replication, which may lead to the stimulation of host immunological responses. The bioavailability of conventional DNA vectors is also compromised due to their larger molecular size. Their circular nature may also impart chromosomal integration, leading to insertional mutagenesis.
Bacterial sequences are excised from DNA minivectors, leaving only the gene of interest (GOI) and necessary eukaryotic expression elements. Our LCC DNA minivectors, or DNA ministrings, are devoid of immunogenic bacterial sequences; therefore improving their bioavailability and GOI expression. In the event of vector integration into the chromosome, the LCC DNA ministring will lethally disrupt the host chromosome, thereby removing the potentially dangerous mutant from the proliferating cell population. Consequently, DNA ministrings offer the benefits of &#39;minicircle&#39; DNA while eliminating the potential for undesirable vector integration events. In comparison to conventional plasmids and their isogenic circular covalently closed (CCC) counterparts, DNA ministrings demonstrate superior bioavailability, transfection efficiency, and cytoplasmic kinetics - they thus require lower amounts of cationic surfactants for effective transfection of target cells.
We have constructed a one-step inducible in vivo system for the production of DNA ministrings in Escherichia coli that is simple to use, rapid, and scalable.

The linear covalently closed (LCC) DNA minivector (DNA ministring) is a non-viral gene delivery vector offering high transfection efficiency and is relevant to any DNA delivery application. The production system is simple, rapid, and scalable in vivo. The following protocol provides visual step-by-step instructions for the production of DNA ministrings.

La produzione di DNA a doppia elica Ministrings

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

Rilevamento di RNA-binding proteins by<em&gt; In Vitro</em&gt; RNA a tendina nella cultura degli adipociti

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

TChIP-Seq: Cellula-tipo-specifico Epigenome profilatura

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...