Extraction of RNA
From SDMRC
Extracting RNA post stimulation and collection
EXAMPLE: Starting w/ four samples (before, 6hr, 12hrs, 24hrs)
- Use the hood in the hallway, remove any stuff that shouldn’t be in there (ie old chemical bottles)
- Work VERY sterile, we have RNAses on us
- Clean hood with ethanol, replace bench paper (new bench paper is kept on top of fridge in hallway)
- 1.7 ml tubes for extracted RNA
- Chloroform [toxic], isopropyl (in Ju Chen’s cabinet- key is kept in his bay)
- Clean gloves with ethanol (do this every time before you touch something), pre-clean tube rack, clean pipettes
- Vortex samples (5 seconds) if frozen thaw first
- Add 200 ul of chloroform
- Don’t touch chloroform to trizol in tube, pipette from a distance. Trash tip in waste bottle *label the waste bottle when finished*
- Vortex for 15 seconds each tube
- Set on bench for 2-3 minutes (set a timer)
- Centrifuge in the cold room (max speed for 15 minutes) This seperates RNA from everything else in the cell. Don’t want to contaminate RNA with cell particles.
- Label the new 1.7 ml tubes *make sure both sets of tubes stay in order the entire time*
- Keep only the upper colorless layer (RNA is here, about 400 ul) and pipette into new corresponding tube. Careful to not disturb the interface between the two layers. Use a 1000 ul and 200 ul tip to take up the 400 ul (maybe 450 ul)
- Change tips between each tube
- If you think you touched the interphase take away the tip and re-centrifuge the tube
- Once the upper layer is in the new tube, add 500ul of isopropyl alcohol to each tube
- Use a 25ml pipette to aliquot some in a tube (instead of the whole bottle)
- Invert tubes three times then set at room temperature for 10 minutes
- Centrifuge the tubes for 15 minutes at max speed (in the cold room) a pellet will form that’s the RNA we will remove from the supernatant
- Remove all the supernatant from the tube after centrifuge (450 ul)
- Do NOT disturb the tiny pellet. Tip the tube 45 degrees and use your glove to see the pellet, keep your eye on the pellet while you draw up liquid,
- Clean RNA with 70% ethanol (in 50 ml tube on bench)
- If you run out create more from the 100% bottle found under the sink (35 ml of 100% ethanol and rest is milli Q water
- Vortex each to clean (few seconds)
- Centrifuge in cold room at max speed for 10 minutes
- After centrifuging remove the ethanol one tube at a time
- Use the bigger pipette to get majority of the liquid and the smaller pipette to get the small amount of liquid remaining
- Close tube after removing liquid so sample doesn’t dry
- After all the tubes have the ethanol removed, leave the tubes open for no more than 5 minutes
- Add 30 ul of milli Q water (on bench in 50 ml tube DEPC) slowly push the water everywhere in the bottom of the tube, different than flushing, cycle the water a few times. Close tube.
- If there is leftover ethanol still in the tube, dry it before adding water
- Re-dissolve pellet on heat block
- Set heat block for 60 degrees celsius
- Remove the timer on the setting (should read “--:--”)
- Remove “mix” and wait for it to heat up
- Place on heat block for 10 minutes
- Place finished tubes in -20 fridge in the lab
