RNA extraction protocol ( Trizol )
Add 1000 µL of Trizol per 50 - 100 mg of parsite tissues in a 2 mL eppendorf tube ( RNAse free ) and place the parasite tissues into their respective , labelled tube .
Add 1 metal bead into each tube and place the tubes into a TissueLyzer .
Run it at 30 Hz for 3 minutes .
Centrifuge at 12,000 x g for 10 minutes at 4°C .
Discard top layer of liquid formed in the eppendorf after the centrifugation ( contains fatty acids ) .
Transfer supernatant into a new and labelled eppendorf tube ( 2 mL ) .
Incubate at room temperature for 5 minutes .
Add 200 µL of chloroform per tube .
Mix vigorously by inverting the tubes up and down for 1 minute .
DO NOT VORTEX !
Centrifuge at 12,000 x g for 15 minutes at 4°C .
Transfer with precaution the aqueous phase ( supernatant ) into a new 2 mL eppendorf tube .
Add 500 µL of RNAse free isopropanol ( 100 % ) and mix thoroughly .
Incubate for 15 minutes at room temperature .
Centrifuge at 12,000 x g for 10 minutes at 4°C .
Discard supernatant .
Add 1000 µL of 75 % ethanol ( kept cold at - 20°C ) per tube .
Vortex thoroughly and centrifuge at 7500g for 5 minutes at 4°C .
Discard supernatant and let the tubes dry out with the cap opened for 5 - 10 minutes .
Add 20 - 50 µL of DEPC treated water into each tube .
Place the tubes into the incubator ( 65°C ) for 1 minute .
Vortex thoroughly and repeat step 19 until RNA is completely dissolved .
