This is a yeast total RNA isolation protocol that works very well and reliably. It takes a couple of hours.
We have also had success with the Zymo Direct-Zol columns which are faster but can be less clean if overloaded.
For general advice working with RNA consult RNA: A Laboratory Manual, from Cold Spring Harbor Press.
Use RNase-free water, reagents, and equipment throughout.
Grow up 5ml liquid yeast culture to OD 2.0 for lots of RNA (or as desired). Spin at 3000g for 4min
at room temperature. Remove supernatant and resuspend pellet in
500ul RNA lysis buffer (10mM Tris-HCl pH8.5, 5mM
EDTA, 2% SDS, 2% stock 2-mercaptoethanol).
Transfer liquid to 1.5ml tube and put on heat block at 83 with
450rpm mixing for 20mins (to disrupt cells and denature proteins).
Spin down for 5mins at 12,000g.
Take supernatant into new tube and add 550 ul of
Phenol pH8. Vortex for 15 mins.
Spin down for 2mins at 12,000g. There should be a lower phenol
phase, a cloudy interphase, and an upper aqueous phase: RNA
partitions mostly into the upper phase. Transfer upper aqueous phase
(roughly 200 ul ) to new 1.5ml tube (labeled
tube N). Add 250 ul RNA lysis buffer to previous
tube (labeled tube P) and vortex tube P for 5mins.
Add 250 ul chloroform to tube P to suck off phenol
from water phase (This completes a phenol:chloroform extraction of
RNA from the original sample). Vortex for 3 mins, then spin 2mins at
12,000g and transfer aqueous phase from tube P to tube N. Discard
A second phenol extraction: add 550 ul phenol to
tube N. Vortex 5mins, spin 2mins at 12,000g.
Transfer aqueous phase to another 1.5ml tube, discard tube N. Add
550 ul phenol:chloroform pH4.5. Vortex 3mins, then
spin 2mins at 12,000g.
Transfer 450 ul aqueous phase to yet another 1.5ml
tube, add 200 ul of 0.6M Sodium acetate pH4.5, mix
by flicking, and spin briefly. Add 600 ul of
phenol:chloroform pH4.5. Vortex 5mins, then spin 2 mins at 12,000g.
Transfer 350 ul aqueous phase to a fresh 1.5ml tube,
add 30 ul 5M Ammonium acetate and 1.1ml ethanol.
Mix well, and precipitate at -80 for 20mins. The sample may be left
for longer, for example overnight, at this point if a pause
Remove the sample from freezer. Cold spin (4) for 15 mins
Thoroughly remove ethanol from pellet, and add 700 ul
80% ethanol. Cold spin (4C) for 2 minutes at 12,000g. Repeat the
ethanol wash and cold spin. This removes all traces of salt,
Dry pellets thoroughly, i.e., pipette off ethanol, removing
all liquid. If necessary, dry with the tubes open on a 37 heat block
(if the RNA sample is pure, this should not degrade the RNA).
Resuspend pellet in 50 ul H2O.
To check the quality of the RNA, pour a 1% agarose gel on RNA-free
equipment, and run using RNA loading dye supplied with a ladder (RiboRuler High Range,
ThermoFisher #SM1821, or ssRNA Ladder, NEB #N0362S). Heat loading dye and
H2O to 95 for 5 minutes, and then cool, to reduce the possibility
of contamination. Mix 1 ul sample, 5 ul H2O, and 6 ul 2X loading dye for
each well. You should see clear ~4000nt and ~2000 nt bands corresponding to
ribosomal RNA (25S/18S), also a blur at ~80nt for tRNA, and good gels
show the 5S and 5.8S rRNAs at ~150nt.
Perform a 2X serial dilution of the sample for more
precise quantification, or run on Agilent Bioanalyzer RNA Nano chip.