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DNA microarray analysis of CfMNPV genes

ABSTRACT: Temporal expression profiles of all 146 ORFs, and their complements, of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) were determined by a modified oligonucleotide based two-channel DNA microarray. Total RNA was isolated at different times post infection from Cf203 insect cells infected with CfMNPV. The cDNA was synthesized, fluorescently labeled with Cy3, and co-hybridized to the microarray chips along with Cy5-labeled CfMNPV viral genomic DNA (vgDNA), which was used as an equimolar reference standard for each gene. From the microarray data, the temporal gene expression profiles could readily be classified into four clusters based on timing of expression. Transcription of some non-coding antisense strands of the CfMNPV genes was also detected, which provided novel insights into viral gene functions.

ORGANISM(S): Choristoneura fumiferana multiple nucleopolyhedrovirus

PROVIDER: GSE14747 | GEO | 2010/01/08

SECONDARY ACCESSION(S): PRJNA112233

REPOSITORIES: GEO

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DNA microarray analysis of CfMNPV genes

Project description:Temporal expression profiles of all 146 ORFs, and their complements, of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) were determined by a modified oligonucleotide based two-channel DNA microarray. Total RNA was isolated at different times post infection from Cf203 insect cells infected with CfMNPV. The cDNA was synthesized, fluorescently labeled with Cy3, and co-hybridized to the microarray chips along with Cy5-labeled CfMNPV viral genomic DNA (vgDNA), which was used as an equimolar reference standard for each gene. From the microarray data, the temporal gene expression profiles could readily be classified into four clusters based on timing of expression. Transcription of some non-coding antisense strands of the CfMNPV genes was also detected, which provided novel insights into viral gene functions. Total RNA was isolated at different times post infection from Cf203 insect cells infected with CfMNPV. The cDNA was synthesized, fluorescently labeled with Cy3, and co-hybridized to the microarray chips along with Cy5-labeled CfMNPV viral genomic DNA (vgDNA), which was used as an equimolar reference standard for each gene.

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Project description:The temporal expression of the 23 CfMNPV genes representing all four temporal classes including its 7 unique genes were determined by a modified oligonucleotide-based two-channel DNA microarray. Transcription of the non-coding (antisense) strands of some of the CfMNPV select genes including the polyhedrin gene was also detected by the array analysis. The expression of four host genes varied several fold throughout virus infection. The microarray chip contained oligonucleotide probes for 23 CfMNPV ORFs and their complements. We first developed a novel normalization protocol using Cy5-labeled CfMNPV viral genomic DNA (vgDNA) as equimolar reference standards for each probe in order to overcome the inherent variability problem of the traditional microarray normalization procedures including use of internal standards. Cy3-labeled cDNA was from total RNA isolated at different times post infection of Cf203 insect cells infected with CfMNPV. Host genes were unsuitable for normalization between microarrays. The DNA microarray results were selectively validated by quantitative RT-PCR (qRT-PCR). The nature of the polyhedrin antisense transcription was further investigated using long range RT-PCR analysis. Keywords: Time course, detection of antisense transcripts, viral genomic DNA normalization

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