Within 24 h of exhibiting these clinical signs, some piglets Everolimus mouse progressively developed indications of central nervous system infection including trembling, excessive salivation, lack of coordination, ataxia, and seizures. Infected piglets sat on their haunches in a

“”dog-like”" position, lay recumbent and paddled, or walked in circles. The appearance of the dissected organs in selected piglets was typical of PRV infection: bleeding in meninges, oedema in the brain, bleeding spots in the lung and on the adenoids [1, 8]. Three strict criteria were imposed for the selection of piglets included in this study: 1) piglets exhibited the typical clinical signs described above; 2) piglets exhibited the expected pathology, especially in brain

and lung; 3) virus isolation, antibody identification or detection of viral antigen-positive tissues were used to confirm the organic infection by PRV, and diseases including Swine Fever (SF), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and other potential bacterial infections which could be clinically and pathologically confused with PRV infection were excluded by viral antigen, antibody identification and PCR detection. Six piglets aged from 2 to 4 days (commercial breed Landrace X Yorkshire) which were infected by PRV but not by the Selleck LGK 974 other tested diseases (see above) and 3 healthy piglets (not infected, and negative for all tests under the strict criteria used above), matched for age and breed from the same farm were used in this experiment. All experiments were carried out in strict accordance Rebamipide with accepted HuaZhong Agricultural University, China and governmental policies. Microarray experimental design Total mRNA samples from the brains and lungs of the 3 normal piglets were pooled for the reference mRNA. Ten independent RNA samples (6 biological replicates for brain and 4 biological replicates of lung) from the 6 infected piglets were paired with the reference sample for hybridization on two-color microarrays. Using a dye-swap configuration, comparing each sample provides technical replicates to adjust for dye bias[9]. A total of 20 slides were used in

this study. RNA purification Total mRNA was prepared using Qiazol reagent (Qiagen, Crawley, West Sussex, UK) following the manufacturer’s instructions. A second purification step was performed immediately post extraction on the isolated total mRNA using the RNeasy Midi kit (Qiagen Inc., Valencia, CA) and each sample was treated with DNase (20 U of grade I DNase; Roche, Lewes, UK) to remove any genomic contamination following the manufacturer’s instructions. With a cut-off of 150 bp, 5S rRNA and tRNAs were removed from the samples by the columns, limiting interference in downstream experiments. RNA concentration and integrity were assessed on the Nanodrop ND-1000 spectrophotometer (Nanodrop, USA) and on the Agilent 2100 bioanalyzer system (Agilent Technologies, Palo Alto, CA), using an RNA 6000 Nano LabChip kit.