Poly A+ RNA was purified from aMPV infected and uninfected control chicken embryo cells. Forward and reverse subtracted cDNA libraries were constructed using the SSH technology. Random clones were picked and nucleotide sequence determined. The identity of the clones was determined by blast search against the public sequence databases. The cDNA inserts of all SSH clones were amplified by PCR using oligonucleotide primers corresponding to the adaptor sequences flanking the cDNA. PCR products were spotted onto poly-L-lysine coated glass slides. Poly A+ RNA extracted from aMPV infected and uninfected control chicken embryo cells was labeled with Cy5 and Cy3 monofunctional dyes, respectively and hybridized with the spotted array. Images of the hybridized arrays were captured with laser confocal scanner and Cy5/Cy3 intensities determined with Quantarray software. The data was normalized based on Arabidopsis thaliana cab gene control spots. Average Cy5/Cy3 ratios were calculated using the Cy5/Cy3 ratios of the replicate spots and the genes exhibiting differential expression were identified. Finally, validation of the differential expression of a selected group of genes was carried out by real-time RT-PCR using SYBR green-based detection. RT-PCR was performed with gene specific primer pairs and was run on ABI 7700 fluorescent sequence detection system.

The genes were classified into various functional groups using Genespring 6.1 software (Silicon Genetics, Redwood City, CA). The percentages represent the % of genes belonging to a particular functional group of the total number of genes with “known” functions. The genes with unknown functions or the sequences that did not have any homology to known sequences in public databases are not shown.