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

Summary

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.

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: aRNA labeling: amino allyl coupling of the dyes

1. Add 1µg of the aRNA samples into 1.5mL microcentrifuge tubes.
2. 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!
3. Add 9µl 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.
4. Add 22µl 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 th....

Discussion

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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CyDye Post-Labeling Reactive Dye Pack	Reagent	GE Healthcare	RPN5661	Contains both Cy3 and Cy5 dyes
NanoDrop ND-1000 UV-VIS spectrophotometer	Other	NanoDrop	ND-1000	Or equivalent spectrophotometer