Generic sample from Equus caballus
Equus caballus
Equine neurological tissue (cortex, cerebellum, midbrain, hindbrain, thalamus, cervical and lumber spinal cord) and spleen were obtained by necropsy from three groups of two horses each (total of 6 individuals) and consisted of 1) naive horses infected with 1 X 105 WNV, intrathecally 2) non naive horses vaccinated for protection against WNV (Prevenile, Intervet-Schering-Plough) and infected with 1 X 105 WNV , intrathecally and 3) horses that were not infected or vaccinated  Tissues were snap frozen and stored at -80oC until RNA extraction.  Total RNA was extracted using Trizol (Guanidium thiocyanate- Invitrogen, Carlsbad, CA) and a traditional phase separation technique.  Concentration and purity of each RNA sample was assessed using spectrophotometric technology (ND-1000, Nanodrop Technologies, Wilmington, DE) and degradation assessed by microfluidics (Agilent 2100 Bio-analyzer, Santa Clara, CA).  The RNA was converted to a normalized cDNA library (SMART PCR cDNA synthesis kit and Advantage 2 PCR kit, Clontech, Mountainview, CA; QIAquick PCR Purification Kit, Qiagen, Valencia, California; and cDNA Normalization Trimmer Kit, Evrogen, Moscow, Russia).