Transcriptional analysis of select CfMNPV genes and some antisense transcripts by an oligonucleotide microarray . Transcriptional analysis of select CfMNPV genes and some antisense transcripts by an oligonucleotide microarray

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 Overall design: Total RNA was isolated from Cf203 cells at 0, 3, 6, 12, 24 and 48 h post infection, as well as from mock-infected Cf203 cells. The Cy3-labeled cDNA derived from 20 μg total RNA was co-hybridized with 10 ng/μl vgDNA to each array at 42ºC. Seven hybridizations in each experiment, two independent hybridization experiments were performed, and therefore 14 samples were analyzed and included in the sample submission including the mock-infected sample.