Name: Vaccinia Virus Infection and Temporal Analysis of Virus Gene Expression: Part 3 (Video) | JoVE Journal
Uploaded: 2009-04-13T00:00:00.000+00:00
Duration: 7 min 35 s
Description: Learn RNA labeling for gene expression analysis using vaccinia virus-infected HeLa cells. This protocol demonstrates amino allyl dye coupling and microarray sample preparation for studying viral and host gene dynamics.

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5 minutes

Whitehead Institute Fellows Funds

Part 1: aRNA labeling: amino allyl coupling of the dyes
<ol>
<li>Add 1&micro;g of the aRNA samples into 1.5mL microcentrifuge tubes.</li>
<li>Vacuum dry the samples on low or no heat until they are completely dry. Cap each tube as soon as it is dry - do not overdry!</li>
<li>Add 9&micro;l coupling buffer to each tube and resuspend the aRNA by gently vortexing for 1 minute. Centrifuge briefly to collect the sample in the bottom of the tube, and then let the sample sit on ice.</li>
<li>Add 22&micro;l high quality DMSO to each tube of Cy3 or Cy5 dye. One tube of dye is enough for 2 samples. The Cy3 dye is for labeling your reference samples, and the Cy5 dye is for labeling your test samples.</li>
<li>Vortex the dyes to mix thoroughly. Keep the dyes in the dark until ready to use. Do not prepare dye earlier than 1 hour before using. Make sure no water gets in the dye/DMSO mix at any point.</li>
<li>Add 11&micro;l of the prepared DMSO/Cy dye to each sample. Mix well by vortexing gently.</li>
<li>Incubate for 30-45 minutes at room temperature. Cover the samples with tinfoil or keep them in a drawer to minimize exposure to light.</li>
<li>After the incubation, add 4.5&micro;l hydroxlyamine to each sample to quench the reaction. Mix well by vortexing gently.</li>
<li>Incubate for another 15 minutes at room temperature. Cover the samples with tinfoil or keep them in a drawer to minimize exposure to light.</li>
</ol>
Part 2: Labeled aRNA clean-up
<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. (Table 1)</li>
<li>Mix well by vortexing.</li>
<li>Aliquote 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. The supernatant should be either a bright pink or a bright blue at this point due to the unincorporated dye molecules.</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 2nd time with 100&micro;l aRNA Wash Solution.</li>
<li>After the 2nd 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 20&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, labeled aRNA samples, and should be either a pale pink or a pale blue.</li>
<li>Carefully transfer the eluted aRNA to a new PCR plate (or PCR tubes).</li>
<li>(Optional step) Check the RNA concentration and the amount of dye in the samples by measuring 1.5&micro;l on a NanoDrop spectrophotometer using the Microarray module.</li>
<li>Immediately hybridize the labeled aRNA onto a microarray platform of your choice, or alternatively, you can store the labeled aRNA at &ndash;80&deg;C until you are ready for hybridization.</li>
</ol>
Table 1 aRNA Binding Mix
<table border="1">
<tbody>
<tr>
<td>Reagent</td>
<td>Amount for 1 reaction</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>
*Mix the RNA binding beads with the bead resuspension solution first **Add the isopropanol and mix well before adding the aRNA binding buffer concentrate.

<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/pubmed?term=((Comparative+analysis+of+viral+gene+expression+programs+during+poxvirus+infection%3A+a+transcriptional+map+of+the+vaccinia+and+monkeypox+genomes%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Comparative analysis of viral gene expression programs during poxvirus infection: a transcriptional map of the vaccinia and monkeypox genomes.</a> PLoS ONE. 3 (7), e2628-e2628 (2008).</li>
<li>Assarsson, E., Greenbaum, J. A., Sundström, 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/pubmed?term=((Kinetic+analysis+of+a+complete+poxvirus+transcriptome+reveals+an+immediate-early+class+of+genes%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci%22%5BJournal%5D)">Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes.</a> Proc. Natl. Acad. Sci. 105 (6), 2140-2145 (2008).</li>
<li>Satheshkumar, P. S., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Poxvirus+transcriptome+analysis%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Poxvirus transcriptome analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 105, E62-E62 (2008).</li>
<li>Assarsson, E., Greenbaum, J. A., Sundström, 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/pubmed?term=((A.+Reply+to+Satheshkumar+and+Moss%3A+Poxvirus+transcriptome+analysis%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">A. Reply to Satheshkumar and Moss: Poxvirus transcriptome analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 105, E63-E64 (2008).</li>
<li>Guerra, S., López-Fernández, L. A., Pascual-Montano, A., Muñoz, M., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+gene+expression+survey+of+vaccinia+virus+infection+of+human+HeLa+cells%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Cellular gene expression survey of vaccinia virus infection of human HeLa cells.</a> J Virol. 77 (11), 6493-6506 (2003).</li>
<li>Guerra, S., López-Fernández, L. A., Conde, R., Pascual-Montano, A., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microarray+analysis+reveals+characteristic+changes+of+host+cell+gene+expression+in+response+to+attenuated+modified+vaccinia+virus+Ankara+infection+of+human+HeLa+cells%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Microarray analysis reveals characteristic changes of host cell gene expression in response to attenuated modified vaccinia virus Ankara infection of human HeLa cells.</a> J Virol. 78 (11), 5820-5824 (2004).</li>
<li>Guerra, S., López-Fernández, L. A., Pascual-Montano, A., Nájera, J. L., Zaballos, A., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Host+response+to+the+attenuated+poxvirus+vector+NYVAC%3A+upregulation+of+apoptotic+genes+and+NF-kappaB-responsive+genes+in+infected+HeLa+cells%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Host response to the attenuated poxvirus vector NYVAC: upregulation of apoptotic genes and NF-kappaB-responsive genes in infected HeLa cells.</a> J Virol. 80 (2), 985-998 (2006).</li>
<li>Guerra, S., Nájera, J. L., González, J. M., López-Fernández, L. A., Climent, N., Gatell, J. M., Gallart, T., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+gene+expression+profiling+after+infection+of+immature+human+monocyte-derived+dendritic+cells+by+the+attenuated+poxvirus+vectors+MVA+and+NYVAC%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Distinct gene expression profiling after infection of immature human monocyte-derived dendritic cells by the attenuated poxvirus vectors MVA and NYVAC.</a> 81 (16), 8707-8721 (2007).</li>
</ol>

The authors have nothing to disclose.

Critical Steps
When performing the amino allyl coupling, it is critical to resuspend the dye in DMSO shortly (less than 1 hour) before coupling and ensure no water gets into the dye/DMSO mix, as it will react with the active group on the dye. Do not overdry the RNA (can be dried down to 1-2uL rather than completely dry), and resuspend well in the coupling buffer. During the coupling reaction, keep the reaction in the dark, with occasional flicking and spin down if desired.
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<table><tbody><tr><td>CyDye Post-Labeling Reactive Dye Pack</td><td>Reagent</td><td>GE Healthcare</td><td>RPN5661</td><td>Contains both Cy3 and Cy5 dyes</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>

vaccinia virus infection &amp; temporal analysis of virus gene expression: part 3

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

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

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

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

Hi, I am Judy Y from the laboratory of Kate Rubins at the Whitehead Institute for Biomedical Research. Here we demonstrate how to label the antisense RNA samples collected from both vaccinia virus infected hela cells and virus by amino all LEO coupling of dyes. We then show how to clean up the labeled A RNA and prepare it to be hybridized for microarray analysis of gene expression.Previous Joe videos highlight how to perform the viral infections total RNA isolation and RNA amplification and amplification steps. So let's get started. To begin a RNA labeling first add one microgram of the A RNA samples into 1.5 milliliter micro centrifuge tubes.Vacuum dry the samples on low or no heat until they are completely dry. Cap each tube as soon as it is dry. It is important to not over dry the samples after they have dried.Add nine microliters coupling buffer to each tube and resuspend the A RNA by gently vortexing for one minute centrifuge briefly to collect the sample on the bottom of the tube and then let the sample sit on ice. Next, add 22 microliters high quality DMSO to each tube of SCI three or S five dye one tube of dye is enough for two samples. The S SI three dies for labeling your reference samples and the SCI five dye is for labeling your test samples.Vortex the dyes to mix thoroughly. Remember to keep the dyes in the dark until ready to use. Do not prepare the dye earlier than one hour before using and make sure no water gets in the dye DMSO mix at any point.Now at 11 microliters of the prepared D-M-S-O-S dye mixture to each sample mixed well by vortexing. Gently incubate for 30 to 45 minutes at room temperature. Cover the samples with tinfoil or keep them in a drawer to minimize exposure to light.After the incubation at 4.5 microliters hydroxy lamine to each sample to quench the reaction mixed well by vortexing. Gently incubate for another 15 minutes of broom temperature. Cover the samples with tinfoil or keep them in a drawer to minimize exposure to light.To clean the label, they RNA vortex, the RNA binding beads. Briefly to obtain an even mixture before use, prepare the A RNA binding mix at room temperature. Mix the binding mix well by vortexing aliquot the A RNA elution buffer into a 1.5 milliliter tube and incubated 50 to 60 degrees Celsius for at least 10 minutes.Add 70 microliters of the A RNA binding mix to each sample and mix well by pipe adding up and down three to four times after mixing, transfer the samples from the PCR plate to a 96 well round bottom plate. Next, add 50 microliters of a hundred percent isopropanol to each sample and mix well by pipetting up and down three to four times. Gently shake the plate on an orbital shaker for at least two minutes.To thoroughly mix the samples. 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. 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.The supernatant should be either a bright pink or a bright blue at this point due to the unincorporated dye molecules. Once the supernatant has been discarded, remove the plate from the magnetic stand. Now add a hundred microliters, a RNA wash solution to each well and shake the plate for one minute on the orbital shaker at moderate speed.Beads may not fully disperse at this step, return the plate to a magnetic stand. To capture the magnetic beads. 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. Remove the plate from the magnetic stand. Repeat the wash a second time with 100 microliters, A RNA wash solution.After the second wash. Dry the beads by shaking the plate for one minute on the orbital shaker at the maximum speed. Do not over dry the samples elute the A RNA from the beads by adding 20 microliters preheated, A RNA elution buffer.To each sample vigorously shake the plate on the orbital shaker for three minutes. Then check to make sure the magnetic beads are fully dispersed. If not, continue shaking.Once the magnetic beads are fully dispersed, move the plate to a magnetic stand to capture the magnetic beads. The supernatant contains the cleaned up labeled A RNA samples and should be either a pale pink or a pale blue. Next, carefully transfer the alluded a RNA to a new PCR plate or PCR tubes.At this point, you can check the RNA concentration and the amount of dye in the samples by measuring 1.5 microliters on a NanoDrop spectrophotometer. Using the microarray module immediately hybridize the labeled A RNA onto a microarray platform of your choice. Or alternatively, you can store the labeled A RNA at negative 80 degrees Celsius until you are ready for hybridization.This is a representative spectrophotometer reading and OD plot generated using the NanoDrop spectrophotometer to measure sample concentration and dye incorporation. You can see the OD peaks at 260 nanometers for calculating the concentration of the samples as well as the two peaks around 532 and 635 nanometers representing the S3 and SCI five dyes respectively. We've just shown you how to fluorescently label amplified immuno allele incorporated RNA samples for microray hybridization.When doing this procedure, it's important to remember to resuspend the dye in DMSO less than one hour before coupling and ensure no water gets into the dye DMSO mix as it will react with the active group on the dye. It is also important to remember not to over dry the RNA if necessary. The samples can be dried down to one to two microliters rather than completely dry, and then resuspended well in the coupling buffer during the coupling reaction.Keep the reactions in the dark with occasional flicking and spin down if desired. So that's it. Thanks for watching and good luck with your experiments.Be sure to watch the other videos in this series where we show how to perform a time course infection with the vaccinia virus and RNA extraction of the samples and the procedure for linear amplification of RNA samples.

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Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

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

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Learn RNA labeling for gene expression analysis using vaccinia virus-infected HeLa cells. This protocol demonstrates amino allyl dye coupling and microarray sample preparation for studying viral and host gene dynamics.

Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 3

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