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
~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.
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.
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.
0.3ml chloroform. Shake the tube
vigorously / 15sec. Let sample
stand at room temperature / 5min.
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.
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
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.
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.
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.
the damp RNA pellet in 50ml DEPC-water by passing the
sample through the pipette tip. Add
RNase inhibitor RNase-Out (Invitrogen’s, 40u/ml)
to the sample.
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
the RNA sample at –80oC.