title: PSLN_K351
organism: Ovis aries
description: Protocols: New Zealand Cheviot sheep of PRNP genotypes VRQ/VRQ, from the DEFRA breeding flock were inoculated subcutaneously with 2 ml of 10% w/v brain homogenate into sites on either side of the neck roughly 10cm anterior to the prescapular lymph node (PSLN). In all experiments frozen lymph node was converted to a frozen tissue powder. Briefly a 100 mg of tissue per sample was placed into a Teflon flask along with steel ball and placed in liquid Nitrogen for 1 min. Homogenised for 60 seconds at 2000 rpm in a Mikro-Dismembrator U (B.Braun Biotech International GmbH). Total RNA was extracted from the frozen tissue powder using the RiboPure Kit (Ambion) including a DNase I digestion. To the frozen tissue powder 1 ml of Tri Reagent was added and then homogenise for a further 15 seconds at 1500rpm. A 200 ul aliquot of chloroform was added to lysate and mixed vigorously by vortexing. The protocol was then followed as per the manufactures instructions. The RNA was eluted in a final volume of 50 ul. Each RNA sample was DNase I (Ambion) treated as per the manufacturers instructions. The quality and quantity of the isolated total RNA was assessed using a RNA LabChip on the Agilent 2100 bioanalyser and a NanoDrop ND-1000 Spectrophotometer (Nanodrop Technologies Inc.). (