This protocol was used in the above Nature Immunology paper
Method Article
RNA isolation
https://doi.org/10.1038/nprot.2007.69
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RNA
This protocol was used in the above Nature Immunology paper
Trizol (GibcoRL, 10296)
100% Isopropanol (Fisher Scientific, A416-500)
70% Ethanol (Sigma E702-3)
DepC Treated water (Ambion 9920)
Chloroform (Fisher Scientific C298-500)
Pellet cells and resuspend in 1 mL of Trizol (I have difficulty isolating quality RNA from less than 1 X 106 primary T cells). 1 mL of Trizol can be used for 1 X 106 to 10 X 106. It is best to brush the pelleted cells along a rack to dislodge them from the bottom of the tube. Sometimes aggregates will still be visible. Do not resuspend.
Incubate at room temperature for 5 minutes
Add 200μL of Chloroform using a barrier pipette tip and shake each sample vigourously
Incubate at room temperature for 3 minutes
Centrifuge at 12000g for 15 minutes at 4°C
Transfer upper aqueous phase into a new RNAase free tube (approximately 0.8-1 mL of volume)
Add 0.5 mL of isopropanol/mL of Trizol used and shake vigourously
Incubate at room temperature for 10 minutes
Centrifuge at 12000g for 15 minutes at 4°C
Pour off supernantant and wash pellet in with 1 mL of 75% ethanol ( do not resuspend pellet rather squirt in ethanol so the pellet becomes dislodged from the bottom of tube)
Centrifuge at 12000g for 5 minutes at room temperature
Pour off supernantant and briefly dry pellet (can use a 10μL pipette or a kimwipe corner to get rid of residual ethanol)
Dissolve pellet in 30μL of DepC treated water and place at 55°C for 5-10 minutes.
Spec and take 2μg for cDNA conversion. Store the rest of the RNA at -80°C.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
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