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.
Highlights
The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells
While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus
The labeled RNA resulting from this protocol can be hybridized to human, viral, or custom microarrays to assess gene expression responses to infected cells in culture
Summary
Shake the bottle to fully resuspend the magnetic cDNA binding beads before use. 3. Add 180μl of cDNA Pure to each sample, and thoroughly mix by pipetting up and down. 4. Continue to mix the samples by gently shaking the plate on an orbital shaker for at least 2 minutes. 8. Add 150μl cDNA Wash Buffer to each well and shake the plate for 1 minute on the orbital shaker at moderate speed. After the 2nd wash, dry the beads by shaking the plate for 2 minutes on the orbital shaker at the maximum speed. Elute the cDNA from the beads by adding 18μl of the preheated nuclease-free water to each sample. Shake the plate for 3 minutes on the orbital shaker, check to make sure the magnetic beads are fully dispersed.
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