Total RNA Isolation from H. contortus Worms

Total RNA was isolated from Adult worm of H. contortus collected from the abomasums of donor goats as described previously [57 ]. The worms were ground using a pre-chilled mortor and pestle. One ml of Trizol (Invitrogen) was added and homogenized for 30 minutes. Then 200μl of Tri-chloromethane was added and the mixture was spun at 12,000rpm for 15 min at 4°C. After that, RNA was precipitated from the supernatant by the addition of 0.25 volumes of isopropyl alcohol per each milliliter of Trizol and incubated at -20°C for 30 min. The RNA was pelleted at 12,000 rpm at 4°C for 10 min. RNA pellet was washed by 70% ethanol, dried andresuspended in DEPC-treated water. RNA integrity was checked by agrose gel electrophoresis and quantified by NanoDrop ND-1000 Spectrophotometer. The RNA solution was used in subsequent cDNA preparation immediately. The cDNA was synthesized by reverse transcription reaction using cDNA Kit (TaKaRa Biotechnology) according to the manufacturer's instructions.