Name: protocol for rnai assays in adult mosquitoes (a. gambiae)
Uploaded: 2007-07-04T00:00:00
Description: Watch this Scientific Journal Video about Protocol for RNAi Assays in Adult Mosquitoes A. gambiae at JoVE.com

<p class="jove_content"> RNAi-medierad geners uttryck i myggor kräver förberedelser och rening av specifika dsRNAs som riktar genen av intresse. För dsRNA syntes, rekommenderar vi att satsen Ambion Megascript som använder sig av en T7 RNA-polymeras-medierad <em>in vitro</em> transkription reaktion. För dsRNA rening rekommenderar vi Qiagen RNeasy kit. Prover på dsRNA bör kvantifieras och justeras till 3 mikrogram / mikroliter i vatten. Andra material du behöver är: spetsig pincett, glasskiva, glas microcapillary nålar, microinjector (v...

<ol>
	<li>Boisson, ., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (2006).</li><li>Blandin, ., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (2002).</li></ol>

<p> Effektivitet av geners uttryck är mycket varierande och beror på ett antal faktorer, inklusive utskrift och protein omsättning priser, och dsRNA upptag effektivitet genom celler och organ. Vi har funnit att tysta börjar så tidigt som en injektion dagen efter och kan pågå upp till 6 dagar efter injektionen. Använd utskrift och protein detektionsmetoder såsom kvantitativa PCR och Western blotting för att validera tysta. </p>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Megascript</td>
<td>kit</td>
<td>Ambion</td>
<td>&nbsp;</td>
<td>for dsRNA synthesis; T7 RNA polymerase-mediated in vitro transcription reaction</td>
</tr>
<tr>
<td>RNeasy</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>&nbsp;</td>
<td>For dsRNA purification</td>
</tr>
<tr>
<td>dsRNA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>should be quantified and adjusted to 3 &#x03BC;g/ &#x03BC;L in water</td>
</tr>
<tr>
<td>fine-tipped forceps</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>glass slide</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>glass microcapillary needles</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>microinjector</td>
<td>&nbsp;</td>
<td>Drummond</td>
<td>Nanoject II</td>
<td>&nbsp;</td>
<tr>
<td>light microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>mounted above a cold block</td>
</tr>
<tr>
<td>Petri dish</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>covered, glass</td>
</tr>
<tr>
<td>paper towels</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10% sucrose</td>
<td>food</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>A. gambiae</td>
<td>Animal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>mosquitos</td>
</tr>		</tbody>
		</table>
		</div>
	

protocol for rnai assays in adult mosquitoes (a. gambiae)

<p class="jove_content">Omvänd genetiska metoder har visat sig extremt användbart för att avgöra vilka gener som underliggande motstånd mot vektor patogener i myggor. Denna video protokoll illustrerar en metod som Dimopoulos labbet att injicera dsRNA i Anopheles gambiae myggor som hyser malariaparasiten. Tekniken manipulera injektionen setup och injicera dsRNA i bröstkorgen illustreras.</p>

Watch this Scientific Journal Video about Protocol for RNAi Assays in Adult Mosquitoes A. gambiae at JoVE.com

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