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-gemedieerde gen-silencing in muggen vereist de voorafgaande bereiding en zuivering van specifieke dsRNA&#39;s die het gen van belang doel. Voor dsRNA synthese, adviseren wij de Ambion Megascript kit die gebruik maakt van een T7 RNA polymerase-mediated <em>in vitro</em> transcriptie reactie maakt. Voor dsRNA zuivering, adviseren wij de Qiagen RNeasy kit. Monsters van dsRNA moeten worden gekwantificeerd en aangepast aan de 3 ug / ul in het water. Andere materialen je nodig hebt onder andere: fijne punt pincet, glazen...

<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> Efficiëntie van gene silencing is zeer variabel en afhankelijk van een aantal factoren, waaronder transcript en eiwit turn-over tarieven en dsRNA opname efficiency door cellen en organen. We hebben ontdekt dat zwijgen zo vroeg een dag na de injectie begint en kan duren tot 6 dagen na de injectie. Gebruik transcript en eiwit detectiemethoden zoals kwantitatieve PCR en Western blotting te zwijgen valideren. </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">Omgekeerde genetische benaderingen hebben bewezen zeer nuttig voor het bepalen van welke genen de basis liggen van resistentie tegen pathogenen in vector muggen. Deze video protocol illustreert een methode die wordt gebruikt door de Dimopoulos lab dsRNA injecteren in Anopheles gambiae muggen die de malariaparasiet haven. De techniek het manipuleren van de injectie installatie en het injecteren van dsRNA in de thorax wordt geïllustreerd.</p>

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

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