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

Summary

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

Abstract

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

Part 1: cDNA synthesis from RNA

1. Before using the Ambion Amino Allyl MessageAmp II kit, add the recommended volume of 100% ethanol to the wash buffers.
2. In PCR reaction tube, add between 100ng to 5µg of total RNA and 1µl of T7 oligo (dT) primer. Bring the volume up to 12µl with nuclease-free water.
3. Incubate samples at 70°C for 10 min in a thermocycler.
4. Remove RNA samples from 70°C and centrifuge briefly. Place on ice.
5. Prepare the 1^st Strand Synthesis master mix and keep at room temperature (with 10% overage for pipetting error). (Table 1)
6. Gently pipette Master....

Discussion

Critical Steps

A second round of amplification is not advised as biases in array data have been observed. Mixing carefully at each enzymatic step (1^st and 2^nd 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 – even deviations as small as 2-3 degrees during IVT in an air hybridization oven or water bath hybridiza.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Amino Allyl MessageAmp II aRNA amplification kit	Reagent	Applied Biosystems	AM1753	For 20 reactions
Amino Allyl MessageAmp II aRNA amplification kit	Reagent	Applied Biosystems	AM1821	For 100 reactions, in a 96-well format
NanoDrop ND-1000 UV-VIS spectrophotometer	Other	NanoDrop	ND-1000	Or equivalent spectrophotometer