Blackface Teladorsagia Solexa
Blackface Teladorsagia Solexa
Lambs were experimentally infected with approximately 2,300 infective L3 Teladorsagia circumcincta larvae three times a week starting at approximately 12 weeks of age and continuing until sacrifice at approximately 27 weeks. Gastric lymph nodes were collected from all lambs, diced and placed in RNAlater (Ambion) at 20C, incubated at 4C overnight and stored at -70C. Total RNA was isolated from tissues using the Qiagen RNeasy Mini kit (www.qiagen.com/literature/render.aspx?id=353). Approximately 30 mg tissue was thawed to 20C and excess RNALater removed by blotting. 600ul Buffer RTL (including 6?l ?-mercaptoethanol) was added and tissues homogenized using a rotor-stator homogenizer. Insoluble material was removed by 3 min centrifugation at 12,000 x g. 600ul of 70% ethanol was added to the cleared supernatant, mixed and immediately applied to an RNAeasy mini column, which was centrifuged at 8,000 x g for 15 s. The columns were washed by the addition of 700 ul Buffer RW1 and centrifugation at 8,000 x g for 15 s. RNeasy column was transferred into a new 2 ml collection tube, 500 ul Buffer RPE was added and centrifuged at 8,000 x g for 15 s, which was repeated. RNA was eluted by the addition of 50 ul RNase-free water directly onto the RNeasy silica-gel membrane and centrifugation 8,000 x g for 1 min. RNA quality and integrity was assessed using a RNA 6000 Nano LabChip on the Agilent 2100 Bioanalyzer and quantified using a NanoDrop ND-1000 spectrophotometer.
Experimental Factor: CLINICALINFORMATION
resistant
Experimental Factor: GROWTHCONDITION
T. circumcincta infected