RNA Isolation from Viral - infected Chlorella NC64A
Grow NC64A to mid - log phase .
Concentrate to 1 x 108 cells / ml .
Use 30 ml concentrated cells per time point ( 3 x 109 cells ~ 91 mg cells wet weight ) .
Infect with virus at m . o . i . = 5 for appropriate time .
Harvest infected cells by centrifugation at 5000 rpm , 5 min . , 4°C .
Discard supernatant and flash freeze pellet in liquid nitrogen .
Store at - 80°C until ready to proceed to step # 8 .
Take 1 to 2 samples from the freezer at a time ( keep on ice ) , break pellets loose with spatula , and add 1 ml TRIZOL reagent to each tube .
Work as quickly as possible to prevent RNA degradation .
Break pellets up with spatula and vortexing .
Pipet into 2 ml microcentrifuge tube ( s ) containing 250 mg of 0.25 - 0.30 mm baked glass beads .
Immediately vortex at highest speed 5 min . , 4°C .
Place at - 80°C overnight .
Repeat steps # 8 - 12 for the remaining samples .
Thaw samples and vortex another 5 minutes .
Add 250 µl chloroform to samples , then vortex briefly .
Let stand at room temp . 3 min .
Centrifuge samples at 12,000 x g , 15 min . , 4°C .
Withdraw the RNA - containing aqueous upper phase and deliver to fresh microcentrifuge tube .
Add 500 µl isopropanol .
Let stand 10 min . at room temp .
Centrifuge at 12,000 x g , 10 min . , 4°C .
Discard supernatant .
Wash pellet with 75 % EtOH with vortexing .
Centrifuge 12,000 x g , 5 min . , 4°C .
Dry pellets in vacuum dessicator 5 - 10 min .
DO NOT DRY COMPLETELY .
DNAse each sample by resuspending each pellet in 250 µl dd - H20 .
Add 250 µl of 2X MOPS , 20mM MgCl2 ( 2X ) , and 100 - 200 U RNAse - free DNAse I .
Incubate 1 - 2 h on ice .
Extract with equal volume of phenol : chloroform ( 500 µl ) .
Centrifuge at 12,000 x g , 5 min . , 4°C .
Withdraw aqueous phase , add NaOAc to 0.3 M .
Add 1 ml isopropanol .
Let stand 10 min . , room temp .
Centrifuge up to 12,000 x g , 10 min . , 4°C .
Discard supernatant .
Wash pellet with 75 % EtOH with vortexing .
Centrifuge 12,000 x g , 5 min . , 4°C .
Dry pellets in vacuum dessicator 5 - 10 min .
Do not dry completely .
Resuspend pellets in 40 - 50 µl water .
