Equipment

-3 mL syringes and 21 g needles (1 per extraction)

-dry ice and crushed ice

-75% EtOH at -20 C (prepared from 95% EtOH and DEPC-treated water)

-DEPC-treated mortar and pestle at -20 C

-RNase-free tips (1 mL, 200 uL, 10 uL)

-RNase-free 1.5 mL microcentrifuge tubes (3 per extraction)

-DEPC-treated forceps and scalpel

-small bottle of isopropanol and chloroform

-DEPC-treated or RNase-free (Sigma) water

-always wear gloves and lab coat

Protocol

1. Cut off a small piece of tissue (max. 100 mg) at -80 C or in liquid nitrogen.
   1. ON DRY ICE:  Grind tissue into a fine powder using the mortar and pestle and scoop into labelled microcentrifuge tube.  
      * Keep forceps, scalpel, mortar and pestle, and tissue samples on dry ice until Trizol is added.
      *Use fume hood for all steps involving Trizol, chloroform and isopropanol (wash area in fume hood with EtOH and put down clean bench cover before you start). 
      *Have bottle in fume hood for chloroform/phenol wastes and sharps container for needles.
   2. In fume hood add 1 mL Trizol (per 100 mg tissue) and homogenize using syringe/needle and keep on ice.
      *For all steps following Trizol addition keep on crushed ice (except for room temp. incubations).
   3. Spin for 15 min at 12 000 g and 4 C.
   4. Decant supernatant into new labelled microcentrifuge tubes. Throw away pellet.
   5. Incubate at room temp. for 5 min.
   6. Add 0.5 mL chloroform per mL Trizol and shake vigorously for 15 s.
   7. Incubate at room temp. for 2-3 min.
   8. Centrifuge for 15 min at 12 000 g and 4 C.
   9. Pipette out the top clear layer and transfer to a new microcentrifuge tube.  Be sure not to pick up any of the middle white layer.
   10. Add 1 mL isopropanol per mL Trizol and vortex to detach pellet.  
       *At this stage the RNA is stable and can be left for a couple hours on ice in the fridge or overnight at -80 C.
   11. Incubate for 10 min at room temp.
   12. Centrifuge for 10 min at 12 000 g and 4 C.  
       *You may want to orient the tubes so that you know where the pellet will form.
   13. Decant the supernatant but DO NOT DISTURB PELLET.  Use a pipettor to remove fluid from around pellet. 
       *Keep all used tips in fume hood to allow chloroform/phenol to evaporate.
   14. Add 1 mL ice-cold EtOH per mL Trizol and vortex to detach pellet.
   15. Centrifuge for 5 min at 7500 g and 4 C.
   16. Decant as before and repeat steps 15-16.
   17. Decant and pulse spin to gather excess EtOH at bottom of tube.  Remove excess with pipettor and/or Kimwipe WITHOUT DISTURBING PELLET.
       *Before air drying pellet twist corner of Kimwipe and use to remove excess EtOH from around pellet and off sides of tube.
   18. 19. Air dry on ice for up to 5 min.  
       *At this stage pellet can be stored at -80 C for up to a year.
   19. Reconstitute in DEPC-treated or RNase-free water (10-50 uL depending on size of pellet).
   20. Incubate for 10 min at 55-60 C tapping tubes occasionally to help dissolve pellet.
   21. Quantify on spec. repeating 2-3 times and use average concentration.
   22. Store at -80 C.
       *Treat with DNase before performing reverse transcription to ensure the product contains only your RNA.