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MacManes Lab RNA Extraction Protocol

Prep

  1. RNAse clean bench, ice bucket, pipettes, etc
  2. Remove sample(s) from -80, take note of which samples you are extracting from. I sometimes even take an iPhone pic for reference.
  3. Place samples in ice to thaw.
  4. Turn on centrifuge to get it cooling down to 4 degrees C.
  5. Make 80% Ethanol if needed. (40ml ETOH, 10ml sterile water) in a 50ml falcon tube.

Extract

  1. Cut up kidney or other tissue using clean razor
  2. Place tissue in tube containing 500uL Trizol (Trizol is in fridge)
  3. Homogenize using mortar and pestle in fume hood (Trizol is toxic and tends to splash a little during homogenization).
  4. Add another 500uL Trizol to being volume up to 1ml, and put Trizol back in fridge.
  5. Incubate homogenizes tissue for 5minutes on hula mixed to allow for complete disassociation of the nucleoprotein complex.
  6. Add 200uL Chlorofom (it's in or under fume hood) - in the fume hood and shake tube for 15 seconds.
  7. Incubate 3 minutes on the hula mixer.
  8. Spin 12,000 RCF for 15 minutes at 4 degrees. Make sure samples are balanced in centrifuge.
  9. Once done, remove the top clear layer and put the clear fluid in a new labelled tube. Get most of the clear fluid, but be sure to not get any of the other stuff.

Precipitate

  1. Add 500uL of Isopropanol (2-propanol) from freezer and incubate for 10 minutes on the hula mixer.
  2. Spin 12,000 RCF for 10 minutes at 4 degrees. Make sure samples are balanced in centrifuge.

Wash RNA

  1. Dump out Isopropanol, with care not to dump out pellet.
  2. Add 1ml of 70%-80% ethanol, which can be found in several places in lab.
  3. Shake the tube, then spin down briefly.
  4. Spin 7,500 RCF for 5 minutes at 4 degrees.
  5. Dump out the ethanol, spin down, then carefully pipette off the remaining ethanol.
  6. Air dry for 5-10 minutes. Be careful not to over dry.
  7. Re-suspend in 50uL sterile water. Mix on hula mixer for 15 minutes, or put in Freezer if not using immediately. If freezer, consider taking 2-3uL out into separate tube for Tapestation quant.

Clean Up

  1. Make sure to put the rest of the tissue samples in the -80 freezer.
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