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"> Silençage génique médiée par ARNi chez les moustiques nécessite une préparation préalable et la purification de dsRNA spécifiques qui ciblent le gène d&#39;intérêt. Pour la synthèse ARNdb, nous recommandons le kit Megascript Ambion qui rend l&#39;utilisation d&#39;un ARN polymérase T7 médié réaction <em>de</em> transcription <em>in vitro.</em> Pour la purification ARNdb, nous recommandons le kit Qiagen RNeasy. Des échantillons d&#39;ARNdb doivent être quantifiées et ajusté à 3 pg / pl dans l&#39;eau...

<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> Efficacité de l&#39;inactivation du gène est très variable et dépend d&#39;un certain nombre de facteurs, dont la transcription et des protéines turn-over des taux, et l&#39;efficacité d&#39;absorption ARNdb par les cellules et les organes. Nous avons trouvé que l&#39;inhibition commence dès le 1er jour après l&#39;injection et peut durer jusqu&#39;à 6 jours après l&#39;injection. Utiliser des méthodes de détection et de la transcription des protéines telles que la PCR quantitative et Western blot pour valider taire. </p>

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

<p class="jove_content">Inverse des approches génétiques se sont avérées extrêmement utiles pour déterminer quels gènes de résistance sous-tendent à des agents pathogènes vecteurs de moustiques. Ce protocole vidéo illustre une méthode utilisée par le laboratoire Dimopoulos à injecter dans les ARNdb Anopheles gambiae moustiques, qui abritent le parasite du paludisme. La technique de manipulation de l&#39;installation d&#39;injection et injection ARNdb dans le thorax est illustré.</p>

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

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