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[DESCRIPTION]
<div class="column">The steps for preparing the lysate are different depending on the starting material. Please ensure you follow the correct procedure for your starting material (see the section <a href="https://www.protocols.io/view/genomic-dna-removal-and-total-rna-purification-fro-f48bqzw?guidelines" target="_blank">Disrupting and Homogenizing Starting Materials</a> and the different lysate preparation protocols <a href="https://www.protocols.io/view/isolate-ii-biofluids-rna-kit-f5kbq4w" target="_blank">here</a>). The subsequent steps detailed in <a href="https://www.protocols.io/view/genomic-dna-removal-and-total-rna-purification-fro-f48bqzw" target="_blank">Genomic DNA removal and total RNA purification</a> are the same in all cases.</div>
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<p>This protocol is for the isolation of integrated viral RNA from mammalian cells growing in culture.</p>

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<p>Ensure Lysis Buffer RX is prepared (see guidelines).</p>
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<p>Ensure that all solutions are at room temperature before use.</p>
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<p>Two types of spin columns are provided with this kit: the <strong>Genomic DNA Removal Column</strong></p>
<p>(blue ring) and the <strong>RNA Column</strong> (black ring). Ensure the correct column is used for each</p>
<p>step of the procedure.</p>
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<p>All centrifugation steps are carried out in a benchtop microcentrifuge at 14,000 x g except</p>
<p>where noted. Perform all centrifugation steps at room temperature.</p>
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<p>It is important to work quickly during this procedure.</p>
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[GUIDELINES]
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<p>A maximum of 3 x 10<sup>6</sup> eukaryotic cells can be used as starting material. A hemocytometer</p>
<p>can be used in conjunction with a microscope to count the number of cells. As a general</p>
<p>guideline, a confluent 3.5 cm plate of HeLa cells will contain 10<sup>6</sup> cells.</p>
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<p>Cell pellets can be stored at -80°C for later use or used directly in the procedure.</p>
<p>Determine the number of cells present before freezing.</p>
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<p>Frozen pellets should be stored for no longer than 2 weeks to ensure that the integrity</p>
<p>of the RNA is not compromised.</p>
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<p>Frozen cell pellets should not be thawed prior to beginning the protocol. Add the Lysis</p>
<p>Buffer RX directly to the frozen cell pellet.</p>
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<div class="column">Additional reagents required: PBS (RNase-free).</div>
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<div class="page" title="Page 12">Please review the Guidelines under <a href="https://www.protocols.io/view/genomic-dna-removal-and-total-rna-purification-fro-f48bqzw?guidelines" target="_blank">Genomic DNA removal and total RNA purification from all types of lysate</a> for other important details.</div>
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[STEPS]
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