- Check the quality of the transcripts by using the Agilent 2100
Bioanalyzer (seeNote 5)(seeSubheading3.4) and proceed to
yeast rRNA reconstruction.
3.3 Yeast Total RNA
Extraction
The protocol for total yeast RNA isolation using hot acid phenol is
adapted from [12].- Transfer yeast cells in 1.5 mL microcentrifuge tubes and pellet
cells by centrifugation at 2500 rpm (600g) for 5 min at room
temperature. Discard the supernatant. - Resuspend the cells in 1 mL of RNase-free water. Centrifuge
for 1 min at full speed at room temperature. Discard the
supernatant. - Resuspend the cell pellet in 400μL of AE buffer.
- Add 40μL of 10% SDS and vortex until the pellet is completely
resuspended. - Add 440μL of acid phenol. Vortex.
- Incubate for 4 min at 65C and then cool rapidly the mixture
on dry ice for 2–3 min. - Centrifuge the samples for 10 min at maximum speed at room
temperature. Transfer carefully the aqueous (upper) phase to a
new 1.5 mL microcentrifuge tube. - Add 420μL of phenol–chloroform–IAA, vortex and centrifuge
for 10 min at full speed at room temperature. - Transfer the aqueous phase to a new 1.5 mL centrifuge tube.
Add 400μL of chloroform. Vortex and centrifuge at full speed
at room temperature for 10 min. - Transfer the aqueous phase to a new 1.5 mL centrifuge tube.
Add 40μL of 3 M NaOAc and 1 mL of 96% ethanol. Place at
80 C for at least 30 min. - Centrifuge for 30 min at 4C at full speed.
- Discard the supernatant and wash pellet with 500μL of 80%
ethanol. - Centrifuge for 10 min at 4C at full speed.
- Discard the supernatant, centrifuge again your samples for a
short spin. - Remove any liquid left.
- Incubate your samples with open lid for 2 min at 37C and
5 min at room temperature. - Resuspend the pellet with 50μL of RNase-free water.
- Quantify yeast total RNA samples by measuring A260nmusing
a UV-spectrophotometer (seeNote 6). Check the quality of
your samples by using the Agilent 2100 Bioanalyzer (seeSub-
heading3.4).
36 Lilia Ayadi et al.