Name: Vaccinia Virus Infection and Temporal Analysis of Virus Gene Expression: Part 2 (Video) | JoVE Journal
Uploaded: 2009-04-10T00:00:00.000+00:00
Duration: 10 min 55 s
Description: Learn vaccinia virus infection of HeLa cells and temporal gene expression analysis using reverse transcription and in vitro amplification. This protocol enables microarray profiling of host and viral RNA dynamics.

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5 minutes

Whitehead Institute Fellows Funds

Part 1: cDNA synthesis from RNA
<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 1st Strand Synthesis master mix and keep at room temperature (with 10% overage for pipetting error). (Table 1)</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 2nd Strand Synthesis master mix on ice (with 10% overage for pipetting error). (Table 2)</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>
Part 2: Double-stranded cDNA clean-up
<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 2nd time with 150&micro;l cDNA Wash Buffer.</li>
<li>After the 2nd 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>
Part 3: in vitro Transcription (IVT)
<ol>
<li>Prepare the IVT master mix at room temperature. (Table 3)</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>
Part 4: aRNA clean-up after IVT
<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 4) 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 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 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>
Table 1: cDNA 1st Strand Synthesis Master Mix
<table border="1" cellpadding="8">
<tbody>
<tr>
<td>Reagent</td>
<td>Amount for 1 reaction</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>
Table 2: cDNA 2nd Strand Synthesis Master Mix
<table border="1" cellpadding="8">
<tbody>
<tr>
<td>Reagent</td>
<td>Amount for 1 reaction</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>
Table 3: IVT Master Mix
<table border="1" cellpadding="8">
<tbody>
<tr>
<td>Reagent</td>
<td>Amount for 1 reaction</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>
Table 4: aRNA Binding Mix
<table border="1" cellpadding="8">
<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
A second round of amplification is not advised as biases in array data have been observed. Mixing carefully at each enzymatic step (1st and 2nd 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...

<table><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>

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

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

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 2 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 Yen from the laboratory of Kate Rubins at the Whitehead Institute for Biomedical Research. After infecting hela cells with vaccinia virus and isolating total RNA, shown in a previous Joe video protocol post and viral RNA can be isolated and amplified using reverse and in vitro transcription, the amplified RNA will be used later in microarray analysis of viral and host gene expression. So let's get started.To begin this c cd NA synthesis from the RNA add the recommended volume of a hundred percent ethanol to the wash buffers before using the ambient amino allele message AMP two kit in a PCR reaction tube, add between 100 nanograms to five micrograms of total RNA and one microliter of T seven oligo primer. Bring the volume up to 12 microliters with nuclease free water. Incubate the samples at 70 degrees Celsius for 10 minutes in a thermocycler.Next, remove the RNA samples from 70 degrees Celsius in centrifuge. Briefly, after centrifugation, place the samples on ice. Prepare the first strand synthesis master mix with a 10%volume overage to cover pipe editing error.Leave it at room temperature gently pipette master mix or flick to mix, and then centrifuge briefly after centrifugation, transfer eight microliters of the master mix to each RNA sample mixed by pipe adding up and down three to four times incubated 42 degrees Celsius for two hours in thermocycler. After incubation centrifuge the samples briefly in place on ice. Immediately proceed to the next step of DS dsd A synthesis.Prepare the second strand synthesis master mix on ice with a 10%volume overrate to compensate for pipetting error gently pipette master mix or flick aix and then centrifuge briefly after centrifugation. Transfer 80 microliters to each sample and gently mixed by pipetting up and down three to four times. Incubate at 16 degrees Celsius for two hours in the thermocycler.After the two hour incubation, proceed with the CDNA cleanup step or freeze immediately a negative 20 degrees Celsius. To begin double stranded CD NA cleanup. First, remove the cDNA pure the refrigerator and allow it to equilibrate to room temperature for 30 minutes before use, shake the bottle to fully resuspend the magnetic CD NA binding beads prior to use aliquot nuclease free water into 1.5 milliliter tube and incubate at 50 to 60 degrees Celsius for at least 10 minutes.During the previous two hour incubation, add 180 microliters of CD NA pure to each sample and thoroughly mixed by pipe adding up and down. Transfer the samples to a 96 well round bottom plate. After the samples have been transferred to a plate, continue to mix the samples by gently shaking the plate on an orbital shaker for at least two minutes.Move the plate to a magnetic stand to capture the magnetic beads. Leave the plate on the stand for approximately six minutes or 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. After the supernatant has been discarded, remove the plate from the magnetic. Stand next at 150 microliters, CDNA wash buffer to each well and shake the plate for one 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. After shaking, 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.Or 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 150 microliters, CDNA wash buffer.After the second wash, dry the beads by shaking the plate for two minutes on the orbital shaker at the maximum speed. It is important to not let the beads over dry. Next elute the CDNA from the beads by adding 18 microliters of the preheated nuclease free water to each sample vigorously.Shake the plate for three minutes on the orbital shaker. 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, carefully transfer the eluded cd NA to a new PCR plate or PCR tubes. You should be recovering approximately 16 microliters of the elucian per.Well proceed directly to the next step or freeze the cd NA at negative 20 degrees Celsius. Prepare the IVT master mix at room temperature. Gently pipette the master mix or flick to mix and centrifuge briefly.After centrifugation, add 24 microliters to each sample and gently mixed by pipetting up and down three to four times incubated, 37 degrees Celsius for 14 hours in a thermocycler. Then hold at four degrees Celsius until ready for the next step, vortex the RNA binding beads briefly to obtain an even mixture before use. Prepare the A RNA binding mix at room temperature.This can be done ahead of time. The prepared binding mix can be stored at room temperature for up to one week. Mix well by vortexing aliquot the A RNA elution buffer into a 1.5 milliliter tube in incubate at 50 to 60 degrees Celsius for at least 10 minutes.Now, add 70 microliters of the A RNA binding mix to each sample and mix well by pipetting up and down three to four times. 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. After the beads have pelleted, carefully aspirate the supernatant with the 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. Next, add 100 microliters a RNA wash solution to each well and shake the plate for one minute on the orbital shaker At moderate speed, the beads do not have to fully disperse. At this step, move 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.Once the supernatant has been removed, move 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.Remember to not over dry the samples elute the A RNA from the beads by adding 40 microliters of the 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 have fully dispersed, move the plate to a magnetic stand to capture the magnetic beads. The super natan contains the cleaned up amino allele, incorporated a RNA.Carefully transfer the eluded a NA to a new PCR plate or PCR tubes. At this point, you can check the RNA concentration of the samples by measuring 1.5 microliters on a NanoDrop spectrophotometer. Immediately continue on to the next step or store the A NA at negative 80 degrees Celsius.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 that doing a second round of amplification is not advised. As biases and array data have been observed, it's also important to remember that mixing carefully at each enzymatic step suggests the first and second strand.CDNA synthesis, or the IVT step is critical to obtaining good amplification yields, as is incubation of each enzymatic step at the appropriate temperature. A PCR cycler with an adjustable heated lid is preferred as even deviations as small as two to three degrees during the in vitro transcription. In an air hybridization oven or water bath hybridization can significantly affect the yield of the amplified product.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 preparation and cleanup of fluorescently labeled RNA.

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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 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.

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

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach.  This protocol describes how to process  tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (γHV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for γHV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

Viral Tracing of Genetically Defined Neural Circuitry

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently tagged proteins simultaneously using the method of transient dominant selection is described here.

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Learn vaccinia virus infection of HeLa cells and temporal gene expression analysis using reverse transcription and in vitro amplification. This protocol enables microarray profiling of host and viral RNA dynamics.

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

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