TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Purpose </strong></p>
<p> </p>
<p>To describe the procedure of RNA extraction on post mortem brain tissue specimen using the Direct-zol™ RNA MiniPrep kit (Zymo). The extracted RNA is used in the diagnosis of lyssavirus infection by the LN34 pan-lyssavirus real-time RT-PCR assay.</p>

[BEFORE_START]
<p><strong>Definitions and Keywords</strong></p>
<p><strong> </strong></p>
<p>BSC – Biological Safety Cabinet</p>
<p>PPE – Personal Protective Equipment</p>
<p>RT-PCR – Reverse Transcription-Polymerase Chain Reaction</p>
<p>RNA – Ribonucleic Acid</p>
<p>RNase – Ribonuclease</p>
<p>Room Temperature – Temperature range of 20°C to 25°C (68°F to 77°F)</p>
<p><strong> </strong></p>
<p><strong>Equipment, Materials and Reagents </strong></p>
<p> </p>
<ol>
<li>Supplies
<ol>
<li>Accessioning labels</li>
<li>Disposable gloves</li>
<li>Sterile polypropylene micro-centrifuge tubes (1.5 and 2 ml, RNase/DNase free)</li>
<li>Aerosol (filter) barrier tips (RNase/DNase free)</li>
<li>Rack for Microcentrifuge tubes</li>
<li>Ice</li>
<li>MagNA Lyser green beads (Roche) (optional)</li>
</ol>
</li>
<li>Equipment and Instruments
<ol>
<li>BSC (Class II)</li>
<li>Pipettes</li>
<li>Vortex</li>
<li>Microcentrifuge</li>
<li>Freezer</li>
<li>Mini beadbeater (optional)</li>
</ol>
</li>
<li>Reagents
<ol>
<li>Direct-zol™ RNA MiniPrep kit (Zymo Research)</li>
<li>Ethanol (95% – 100%, molecular grade recommended)</li>
</ol>
</li>
<li>Acceptable surface decontaminants
<ol>
<li>RNase Away® (Fisher Scientific)</li>
<li>70% Ethanol</li>
</ol>
</li>
<li>Reagent Preparation
<ul>
<li>Add ethanol (95-100%) to Direct-zol RNA PreWash concentrate and RNA Wash Buffer concentrate as advised by the manufacturer.</li>
</ul>
</li>
</ol>
<p> </p>
<p><strong>Quality Control and Corrective Action </strong></p>
<p><strong> </strong></p>
<ul>
<li>All issues, comments, concerns, or deviations from the protocol are to be documented and brought to the attention of the lab supervisor or technical supervisor immediately if they are thought to affect the quality of the end product in any aspect.</li>
<li>Temperature levels are to be monitored on applicable equipment to ensure incubators, refrigerators, and freezers stay within criteria set for acceptable temperature range.</li>
<li>Specimen for RNA extraction will be assigned based on the test ordered and/or the recommendations of the technical supervisor or lab supervisor.</li>
<li>All samples are to have unique identifiers and corresponding accession labels provided in advance of starting the RNA extraction procedure.</li>
</ul>

[GUIDELINES]
<p><strong>Purpose </strong></p>
<p> </p>
<p>To describe the procedure of RNA extraction on post mortem brain tissue specimen using the Direct-zol™ RNA MiniPrep kit (Zymo). The extracted RNA is used in the diagnosis of lyssavirus infection by the LN34 pan-lyssavirus real-time RT-PCR assay.</p>
<p> </p>
<p><strong>Responsibility </strong></p>
<p><strong> </strong></p>
<p>Technician will perform the procedure according to the protocol. Once the RNA extraction procedure is complete, the technician is to clean the work area, discard any biohazardous waste, and return the samples to storage.</p>
<p><strong> </strong></p>
<p><strong>Specimens </strong></p>
<p><strong> </strong></p>
<p>Homogenized brain tissue representing a full cross section of brain stem in TRIzol reagent</p>

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