Total RNA Isolation from Triphysaria tissues (root tips, roots, shoots):

(adapted from the Invitrogen’s protocol of Trizol reagent)

 

NOTE: Always wear clean gloves and change gloves often.  All microcentrifuge tubes and pipette tips should be RNase-free.  Clean the pipettors (disassembled parts) with 2% SDS (prepared with DEPC-water), rinse them with DEPC-water and air-dry in a sterile hood; reassemble the pipettor parts in the sterile hood with clean gloves.  Clean the work bench thoroughly.  Clean the tube rack the same way as done for the pipettors.

 

  1. Grind ~100mg frozen tissues in liquid nitrogen in a chilled mortar with a chilled pestle to very fine powder.  Use a chilled spatula to transfer the ground tissue to a 2ml microcentrifuge tube placed on ice.
  2. Add 1.5ml Trizol reagent.  Mix the tissue with Trizol reagent by vigorously inverting the tube.  If processing multiple samples, leave the sample on ice while carrying out steps 1 and 2 for the other samples.
  3. Pellet the insoluble materials (cell membranes, polysaccharides, high molecular weight DNA) from the homogenate by centrifuging the sample at 25,000g (14,000rpm in the Eppendorf’s 5471R centrifuge) / 4oC / 10min.  Transfer the RNA-containing supernatant to a new tube.  Leave the sample at room temperature / 5min to dissociate proteins from RNAs.
  4. Add 0.3ml chloroform.  Shake the tube vigorously / 15sec.  Let sample stand at room temperature / 5min.
  5. Separate the organic phase from the aqueous phase by centrifuging the sample at 25,000g / 4oC / 15min.  Avoiding jerky movement to prevent re-mixing the 2 phases, carefully transfer ~ 0.9ml upper aqueous phase to a new tube.
  6. Precipitate RNA by adding 0.75ml 100% EtOH.  Mix the sample with EtOH and incubate on ice / 15min.  Pellet the precipitated RNA by centrifuging at 12,000g (11,500rpm in the Eppendorf’s 5471R centrifuge) / 4oC / 10min.
  7. Decant the supernatant.  Wash the RNA pellet with 1.5ml 75% EtOH (prepared with DEPC-water).  Centrifuge at 7,500g (8,500rpm in the Eppendorf’s 5471R centrifuge) / 4oC / 5min.
  8. Carefully decant the supernatant to a new tube.  Check the bottom of the old tube for the presence of the transparent RNA pellet before discarding the new tube containing the washing 75% EtOH.
  9. Centrifuge the tube containing the RNA pellet at 12,000g / 4oC / 1min to collect the residual EtOH at the tube bottom.  Remove this residual EtOH from the tube with pipette tip.
  10. Dissolve the damp RNA pellet in 50ml DEPC-water by passing the sample through the pipette tip.  Add 1ml RNase inhibitor RNase-Out (Invitrogen’s, 40u/ml) to the sample.
  11. Check the RNA integrity and yield by running 1ml RNA sample with 1ml 2x loading dye (0.08% w/v bromophenol blue, 14% w/v sucrose) in non-denaturing 1xTAE agarose gel.  (It’s unnecessary to include the denaturing agents in the RNA sample for running in non-denaturing gel.)
  12. Store the RNA sample at –80oC.