RNA-isolation (TRIZOL method)¶
Contributed by Ian Chin-Sang, Queens University, ON, Canada
Isolation of RNA from whole worms using TRIZOL reagent (Gibco-BRL)
- Wash worms from a 60 mm plate with 1 ml DEPC-treated water.
- Spin down worms at 4000xg in microfuge for 1 minute.
- Remove excess water and add 1 ml Trizol reagent. Vortex and invert tube. Let sit at room termerature for 10 minutes (should see stringy-like material).
- Spin tubes in microfuge at 14K for 10 minutes at 4° C.
- Remove supernatant into fresh RNAse free eppendorf tube and add 200 µl of chloroform.
- Vortex for 15 seconds and let sit at room temperature for 3 minutes.
- Spin in microfuge at 12K for 15 minutes at 4° C.
- Carefully remove the top layer (clear) into a new RNAse free eppendorf tube and add 500 µl of Isopropanol. Invert to mix. Let sit for 10 minutes at room temperature.
- Spin in microfuge at 12K for 10 minutes at 4° C. (RNA will be seen as a nice white pellet).
- Wash pellet with 100 µl of 75% ethanol (made by diluting into DEPC-treated water).
- Spin in microfuge at 7500 xg for 5 minutes at 4° C.
- Remove supernatant and air dry pellet for 10 minutes.
- Dissolve pellet in 25 µl of DEPC-treated water. Heat for 10 minutes at 60° C to help dissolve RNA.
This method is based, with permission, on an original protocol available here.