- Report each area for 18S and 25S rRNA indicated for each lane
under the electropherogram and compare them between the
lanes. - On this basis, adjust the quantity for each in vitro and purified
rRNA compared to the control lane. - When these in vitro and purified rRNA reconstituted mixes are
ready to use (seeNote 14), combine them in different stoichio-
metric proportions to obtain 1:0; 0.75:0.25; 0.5:0.5;
0.25:0.75, and 0:1 mixes. - Proceed with alkaline hydrolysis.
3.6 Alkaline
Hydrolysis and RNA
Fragmentation Quality
Control
3.6.1 Alkaline Hydrolysis
- Prepare one 1.5 mL tube per sample to be analyzed containing
10 μL of 3 M NaOAc, 1μL of GlycoBlue, and 1 mL of 96%
ethanol for subsequent precipitation of the sample (“precipita-
tion tubes,” store at 20 C until further use). - Dilute your RNA samples to a concentration of 10 ng/μL with
RNase-free water. - To individual PCR tubes, add 5μL of each of your diluted
RNA samples, keep on ice until further use. - Add 5μL of sodium bicarbonate buffer and mix by pipetting up
and down. - Incubate in a thermal cycler preheated at 95C. Start a timer
and incubate for 10 min (seeNote 15). - Proceed with the next sample every 30 s.
- Stop each reaction after 10 min at 95C by spinning down the
PCR microcentrifuge tube and add the whole sample into the
corresponding 1.5 mL precipitation tube fromstep 1. - Mix by inverting the tubes several times and throw them into
liquid nitrogen. - Recover your samples from the liquid nitrogen and centrifuge
them for 30 min at 4C at full speed in a microcentrifuge. - Remove the supernatant and make sure not to lose the pellet.
- Wash with 600μL of 80% ethanol.
- Centrifuge your samples for 10 min at 4C at full speed.
- Remove the supernatant.
- Centrifuge 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 20μL of RNase-free water.
40 Lilia Ayadi et al.