Development of an oligonucleotide-based DNA microarray for transcriptional analysis of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) genes.
A modified oligonucleotide-based two-channel DNA microarray was developed for characterization of temporal expression profiles of select Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) ORFs including its 7 unique ORFs. The microarray chip contained oligonucleotide probes for 23 CfMNPV ORFs and their complements as well as five host genes. Total RNA was isolated at different times post infection from Cf203 insect cells infected with CfMNPV. The cDNA was synthesized, fluorescent labelled with Cy3, and co-hybridized to the microarray chips along with Cy5-labelled viral genomic DNA, which served as equimolar reference standards for each probe. Transcription of the 7 CfMNPV unique ORFs was detected using DNA microarray analysis and their temporal expression profiles suggest that they are functional genes. The expression levels of three host genes varied throughout virus infection and therefore were unsuitable for normalization between microarrays. The DNA microarray results were compared to quantitative RT-PCR (qRT-PCR). Transcription of the non-coding (antisense) strands of some of the CfMNPV select genes including the polyhedrin gene, was also detected by array analysis and confirmed by qRT-PCR. The polyhedrin antisense transcript, based on long-range RT-PCR analysis, appeared to be a read-through product of an adjacent ORF in the same orientation as the antisense transcript.
Yang
Dan-Hui
DH
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ont. N1G 2W1, Canada.
Barari
Mehrnoosh
M
Arif
Basil M
BM
Krell
Peter J
PJ
D000818:Animals; D020869:Gene Expression Profiling / Q000379:methods*; D017344:Genes, Insect; D005814:Genes, Viral; D009036:Moths / Q000235:genetics / Q000821:virology*; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016366:Open Reading Frames; D012313:RNA / Q000032:analysis; D016372:RNA, Antisense / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016372:RNA, Antisense; D012333:RNA, Messenger; D012367:RNA, Viral; D012313:RNA