Total RNA was isolated from pooled guts and salivary glands of I. scapularis female ticks injected with subolesin dsRNA or injection buffer at 9 dpi (8 days of feeding) using TriReagent (Sigma) according to manufacturer’s instructions. SSH was performed at Evrogen JCS (Moscow, Russia) as described previously (Naranjo et al., 2006; de la Fuente et al., 2007b). Tester and driver RNAs were subtracted in both directions to construct two SSH libraries enriched for differentially expressed cDNAs in subolesin dsRNA-injected (reverse-subtracted) and saline-injected (forward-subtracted) ticks. Tick cDNA fragments (384 from each of the reverse and forward subtracted SSH libraries) were amplified by PCR using pAL-16 vector-specific primers, purified (MultiScreen PCR plates, Millipore, Billerica, MA, USA). Eight pools of 12 clones each from an unsubtracted I. scapularis cDNA library (de la Fuente et al., 2005a) and subolesin cDNA were also arrayed and used to validate normalization. The PCR products were purified using Millipore Multi-screen plates and resuspended in 3x SSC to a concentration of about 500ng/ul. Each probe was then arrayed in quadruplicate onto gamma amino propyl silane coated GAPS II slides (Corning, Lowell, MA, USA).at room temperature and 50% relative humidity using a GeneMachines Omnigrid 100 (Genomic Solutions; Ann Arbor, MI) robot fitted with 4x2 TeleChem SMP4 pins. Printed slides were baked at 80°C for 2 hours. Slides with immobilized probes were then stored in slide holders and placed in a desiccator at room temperature until use.
