<p class="jove_content">Whitehead Institute Fellows Funds</p>

<p class="jove_title">Part 1: cDNA synthesis from RNA</p>
<ol>
<li>Before using the Ambion Amino Allyl MessageAmp II kit, add the recommended volume of 100% ethanol to the wash buffers.</li>
<li>In PCR reaction tube, add between 100ng to 5&micro;g of total RNA and 1&micro;l of T7 oligo (dT) primer.  Bring the volume up to 12&micro;l with nuclease-free water.</li>
<li>Incubate samples at 70&deg;C for 10 min in a thermocycler.</li>
<li>Remove RNA samples from 70&deg;C and centrifuge briefly.  Place on ice.</li>
<li>Prepare the 1<sup>st</sup> Strand Synthesis master mix and keep at room temperature (with 10% overage for pipetting error). <strong>(Table 1)</strong></li>
<li>Gently pipette Master Mix or flick to mix, and then centrifuge briefly.</li>
<li>Transfer 8&micro;l of the Master Mix to each RNA sample.  Mix by pipetting up and down 3-4 times.</li>
<li>Incubate at 42&deg;C for 2 hours in thermocycler.</li>
<li>Centrifuge samples briefly and place on ice.  Immediately proceed to the next step of dsDNA synthesis.</li>
<li>Prepare the 2<sup>nd</sup> Strand Synthesis master mix on ice (with 10% overage for pipetting error). <strong>(Table 2)</strong></li>
<li>Gently pipette Master Mix or flick to mix, and then centrifuge briefly.</li>
<li>Transfer 80&micro;l to each sample and gently mix by pipetting up and down 3-4 times.</li>
<li>Incubate at 16&deg;C for 2 hours in thermocycler.</li>
<li>After the 2 hour incubation, proceed with the cDNA clean-up step or freeze immediately at &ndash;20&deg;C.</li>
</ol>
<p class="jove_title">Part 2: Double-stranded cDNA clean-up</p>
<ol>
<li>Remove the cDNA Pure from the refrigerator and allow it to equilibrate to room temp for 30 minutes before use.  Shake the bottle to fully resuspend the magnetic cDNA binding beads before use.</li>
<li>Aliquot nuclease-free water into a 1.5mL tube and incubate at 50-60&deg;C for at least 10 minutes during the previous 2hr incubation.</li>
<li>Add 180&micro;l of cDNA Pure to each sample, and thoroughly mix by pipetting up and down.  Transfer the samples to a 96-well round-bottom plate.</li>
<li>Continue to mix the samples by gently shaking the plate on an orbital shaker for at least 2 minutes.</li>
<li>Move the plate to a magnetic stand to capture the magnetic beads.  Leave plate on the stand for approximately 6 minutes, or until the mixture becomes transparent and the binding beads have pelleted.</li>
<li>Carefully aspirate the supernatant with a vacuum aspirator without disturbing the magnetic beads.  Alternatively, carefully remove the supernatant with a pipette and discard the supernatant.</li>
<li>Remove the plate from the magnetic stand.</li>
<li>Add 150&micro;l cDNA Wash Buffer to each well and shake the plate for 1 minute on the orbital shaker at moderate speed.  Beads will NOT disperse at this step, due to the low surface tension of the wash buffer.</li>
<li>Move the plate to a magnetic stand to capture the magnetic beads.</li>
<li>Carefully aspirate the supernatant with a vacuum aspirator without disturbing the magnetic beads.  Alternatively, carefully remove the supernatant with a pipette and discard the supernatant.</li>
<li>Remove the plate from the magnetic stand.</li>
<li>Repeat the wash a 2<sup>nd</sup> time with 150&micro;l cDNA Wash Buffer.</li>
<li>After the 2<sup>nd</sup> wash, dry the beads by shaking the plate for 2 minutes on the orbital shaker at the maximum speed.  Do not overdry!</li>
<li>Elute the cDNA from the beads by adding 18&micro;l of the preheated nuclease-free water to each sample.</li>
<li>Vigorously shake the plate for 3 minutes on the orbital shaker, then check to make sure the magnetic beads are fully dispersed.  If not, continue shaking.</li>
<li>Once the magnetic beads have fully dispersed, move the plate to a magnetic stand to capture the magnetic beads.</li>
<li>Carefully transfer the eluted cDNA (~16&micro;l) to a new PCR plate (or PCR tubes).</li>
<li>Proceed directly to the next step, or freeze the cDNA at -20&deg;C. </li>
</ol>
<p class="jove_title">Part 3: <em>in vitro</em> Transcription (IVT)</p>
<ol>
<li>Prepare the IVT master mix at room temperature. <strong>(Table 3)</strong></li>
<li>Gently pipette the Master Mix or flick to mix, and centrifuge briefly.</li>
<li>Add 24&micro;l to each sample, and gently mix by pipetting up and down 3-4 times.</li>
<li>Incubate at 37&deg;C for 14 hours in a thermocycler, then hold at 4&deg;C until ready for the next step.</li>
</ol>
<p class="jove_title">Part 4: aRNA clean-up after IVT</p>
<ol>
<li>Vortex the RNA binding beads briefly to obtain an even mixture before use.</li>
<li>Prepare the aRNA Binding Mix at room temperature. <strong>(Table 4)</strong> This can be done ahead of time--the prepared binding mix can be stored at room temperature for up to one week.</li>
<li>Mix well by vortexing.</li>
<li>Aliquot the aRNA Elution Buffer into a 1.5mL tube and incubate at 50-60&deg;C for at least 10 minutes.</li>
<li>Add 70&micro;l of the aRNA binding mix to each sample and mix well by pipetting up and down 3-4 times.</li>
<li>Transfer the samples from the PCR plate to a 96-well round-bottom plate.</li>
<li>Add 50&micro;l of 100% isopropanol to each sample and mix well by pipetting up and down 3-4 times.</li>
<li>Gently shake the plate on an orbital shaker for at least 2 minutes to thoroughly mix the samples.</li>
<li>Move the plate to a magnetic stand to capture the magnetic beads.  Leave the plate on the stand until the mixture becomes transparent and the binding beads have pelleted.</li>
<li>Carefully aspirate the supernatant with a vacuum aspirator without disturbing the magnetic beads.  Alternatively, carefully remove the supernatant with a pipette and discard the supernatant.</li>
<li>Remove the plate from the magnetic stand.</li>
<li>Add 100&micro;l aRNA Wash Solution to each well and shake the plate for 1 minute on the orbital shaker at moderate speed.  Beads may not fully disperse at this step.</li>
<li>Move the plate to a magnetic stand to capture the magnetic beads.</li>
<li>Carefully aspirate the supernatant with a vacuum aspirator without disturbing the magnetic beads.  Alternatively, carefully remove the supernatant with a pipette and discard the supernatant.</li>
<li>Remove the plate from the magnetic stand.</li>
<li>Repeat the wash a 2<sup>nd</sup> time with 100&micro;l aRNA Wash Solution.</li>
<li>After the 2<sup>nd</sup> wash, dry the beads by shaking the plate for 1 minute on the orbital shaker at the maximum speed.  Do not overdry the samples!</li>
<li>Elute the aRNA from the beads by adding 40&micro;l preheated aRNA Elution Buffer to each sample.</li>
<li>Vigorously shake the plate on the orbital shaker for 3 minutes, then check to make sure the magnetic beads are fully dispersed.  If not, continue shaking.</li>
<li>Once the magnetic beads have fully dispersed, move the plate to a magnetic stand to capture the magnetic beads.  The supernatant contains the cleaned-up amino-allyl incorporated aRNA.</li>
<li>Carefully transfer the eluted aRNA to a new PCR plate (or PCR tubes).</li>
<li>(Optional step) Check the RNA concentration of the samples by measuring 1.5&micro;l on a NanoDrop spectrophotometer.</li>
<li>Immediately continue onto the next step, or store the aRNA at &ndash;80&deg;C.</li>
</ol>
<p class="jove_title">Table 1: cDNA 1<sup>st</sup> Strand Synthesis Master Mix</p>
<table border="1" cellpadding="8">
<tbody>
<tr>
<td><strong>Reagent</strong></td>
<td><strong>Amount for 1 reaction</strong></td>
</tr>
<tr>
<td>10X First Strand Buffer</td>
<td>2 &micro;l</td>
</tr>
<tr>
<td>dNTP Mix</td>
<td>4 &micro;l</td>
</tr>
<tr>
<td>Ribonuclease Inhibitor</td>
<td>1 &micro;l</td>
</tr>
<tr>
<td>Array Script</td>
<td>1 &micro;l</td>
</tr>
</tbody>
</table>
<p class="jove_title">Table 2: cDNA 2<sup>nd</sup> Strand Synthesis Master Mix</p>
<table border="1" cellpadding="8">
<tbody>
<tr>
<td><strong>Reagent</strong></td>
<td><strong>Amount for 1 reaction</strong></td>
</tr>
<tr>
<td>Nuclease free water</td>
<td>63 &micro;l</td>
</tr>
<tr>
<td>10X Second Strand Buffer</td>
<td>10 &micro;l</td>
</tr>
<tr>
<td>dNTP Mix</td>
<td>4 &micro;l</td>
</tr>
<tr>
<td>DNA Polymerase</td>
<td>2 &micro;l</td>
</tr>
<tr>
<td>RNase H</td>
<td>1 &micro;l</td>
</tr>
</tbody>
</table>
<p class="jove_title">Table 3: IVT Master Mix</p>
<table border="1" cellpadding="8">
<tbody>
<tr>
<td><strong>Reagent</strong></td>
<td><strong>Amount for 1 reaction</strong></td>
</tr>
<tr>
<td>aaUTP solution (75 mM)</td>
<td>2 &micro;l</td>
</tr>
<tr>
<td>ATP, CTP, GTP mix (25 mM)</td>
<td>12 &micro;l</td>
</tr>
<tr>
<td>T7 UTP solution (75 mM)</td>
<td>2 &micro;l</td>
</tr>
<tr>
<td>T7 10X Reaction Buffer</td>
<td>4 &micro;l</td>
</tr>
<tr>
<td>T7 Enzyme Mix</td>
<td>4 &micro;l</td>
</tr>
</tbody>
</table>
<p class="jove_title">Table 4: aRNA Binding Mix</p>
<table border="1" cellpadding="8">
<tbody>
<tr>
<td><strong>Reagent</strong></td>
<td><strong>Amount for 1 reaction</strong></td>
</tr>
<tr>
<td>RNA Binding Beads*</td>
<td>10 &micro;l</td>
</tr>
<tr>
<td>Bead Resuspension Solution*</td>
<td>4 &micro;l</td>
</tr>
<tr>
<td>100% isopropanol**</td>
<td>6 &micro;l</td>
</tr>
<tr>
<td>aRNA Binding Buffer Concentrate</td>
<td>50 &micro;l</td>
</tr>
</tbody>
</table>
<p class="jove_content">* Mix the RNA binding beads with the bead resuspension solution first.<br />** Add the isopropanol and mix well before adding the aRNA binding buffer concentrate.</p>

<ol>
	<li>Rubins, K. H., Hensley, L. E., Bell, G. W., Wang, C., Lefkowitz, E. J., Brown, P. O., Relman, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+viral+gene+expression+programs+during+poxvirus+infection:+a+transcriptional+map+of+the+vaccinia+and+monkeypox+genomes.">Comparative analysis of viral gene expression programs during poxvirus infection: a transcriptional map of the vaccinia and monkeypox genomes.</a> <em style='font-style: italic'>PLoS ONE</em>. <b>3</b> (7), e2628-e2628 (2008).</li><li>Assarsson, E., Greenbaum, J. A., Sundstr&#246;m, M., Schaffer, L., Hammond, J. A., Pasquetto, V., Oseroff, C., Hendrickson, R. C., Lefkowitz, E. J., Tscharke, D. C., Sidney, J., Grey, H. M., Head, S. R., Peters, B., Sette, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+a+complete+poxvirus+transcriptome+reveals+an+immediate-early+class+of+genes.">Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes.</a> <em style='font-style: italic'>Proc. Natl. Acad. Sci</em>. <b>105</b> (6), 2140-2145 (2008).</li><li>Satheshkumar, P. S., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poxvirus+transcriptome+analysis.">Poxvirus transcriptome analysis.</a> <em style='font-style: italic'>Proc. Natl. Acad. Sci. U.S.A</em>. <b>105</b>, E62-E62 (2008).</li><li>Assarsson, E., Greenbaum, J. A., Sundstr&#246;m, M., Schaffer, L., Hammond, J. A., Pasquetto, V., Oseroff, C., Hendrickson, R. C., Lefkowitz, E. J., Tscharke, D. C., Sidney, J., Grey, H. M., Head, S. R., Peters, B., Sette, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A.+Reply+to+Satheshkumar+and+Moss:+Poxvirus+transcriptome+analysis.">A. Reply to Satheshkumar and Moss: Poxvirus transcriptome analysis.</a> <em style='font-style: italic'>Proc. Natl. Acad. Sci. U.S.A</em>. <b>105</b>, E63-E64 (2008).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Pascual-Montano, A., Mu&#241;oz, M., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+gene+expression+survey+of+vaccinia+virus+infection+of+human+HeLa+cells.">Cellular gene expression survey of vaccinia virus infection of human HeLa cells.</a> <em style='font-style: italic'>J Virol</em>. <b>77</b> (11), 6493-6506 (2003).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Conde, R., Pascual-Montano, A., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+reveals+characteristic+changes+of+host+cell+gene+expression+in+response+to+attenuated+modified+vaccinia+virus+Ankara+infection+of+human+HeLa+cells.">Microarray analysis reveals characteristic changes of host cell gene expression in response to attenuated modified vaccinia virus Ankara infection of human HeLa cells.</a> <em style='font-style: italic'>J Virol</em>. <b>78</b> (11), 5820-5824 (2004).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Pascual-Montano, A., N&#225;jera, J. L., Zaballos, A., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+response+to+the+attenuated+poxvirus+vector+NYVAC:+upregulation+of+apoptotic+genes+and+NF-kappaB-responsive+genes+in+infected+HeLa+cells.">Host response to the attenuated poxvirus vector NYVAC: upregulation of apoptotic genes and NF-kappaB-responsive genes in infected HeLa cells.</a> <em style='font-style: italic'>J Virol</em>. <b>80</b> (2), 985-998 (2006).</li><li>Guerra, S., N&#225;jera, J. L., Gonz&#225;lez, J. M., L&#243;pez-Fern&#225;ndez, L. A., Climent, N., Gatell, J. M., Gallart, T., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+gene+expression+profiling+after+infection+of+immature+human+monocyte-derived+dendritic+cells+by+the+attenuated+poxvirus+vectors+MVA+and+NYVAC.">Distinct gene expression profiling after infection of immature human monocyte-derived dendritic cells by the attenuated poxvirus vectors MVA and NYVAC.</a> <b>81</b> (16), 8707-8721 (2007).</li></ol>

<p class="jove_content">The authors have nothing to disclose.</p>

<p class="jove_title">Critical Steps</p>
<p class="jove_content">A second round of amplification is not advised as biases in array data have been observed.  Mixing carefully at each enzymatic step (1<sup>st</sup> and 2<sup>nd</sup> strand cDNA synthesis, IVT) is critical to obtaining good amplification yields, as is incubation of each enzymatic step at the appropriate temperature.  A PCR cycler with adjustable heated lid is preferred &ndash; even deviations as small as 2-3 degrees during IVT in an air hybridization oven or water bath hybridiza...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>Amino Allyl MessageAmp II aRNA amplification kit</td>
<td>Reagent</td>
<td>Applied Biosystems</td>
<td>AM1753</td>
<td>For 20 reactions</td>
</tr>
<tr>
<td>Amino Allyl MessageAmp II aRNA amplification kit</td>
<td>Reagent</td>
<td>Applied Biosystems</td>
<td>AM1821</td>
<td>For 100 reactions, in a 96-well format</td>
</tr>
<tr>
<td>NanoDrop ND-1000 UV-VIS spectrophotometer</td>
<td>Other</td>
<td>NanoDrop</td>
<td>ND-1000</td>
<td>Or equivalent spectrophotometer</td>
</tr>		</tbody>
		</table>
		</div>
	

vaccinia virus infection &amp; temporal analysis of virus gene expression: part 2

The family <em>Poxviridae</em> consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of the <em>orthopox</em> genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family.  Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late.  While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus.  Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells.  Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection.  Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.   

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Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

<p class="jove_content">Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.</p>

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

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7.9K Views.  MIT - Massachusetts Institute of Technology. Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.