Characterization of RNA - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 131

most practical technique that results in high yield and quality is
to invert the tubes 8-10 times and immediately place the tubes in
a −80°C freezer until RNA isolation and then allow the blood
tubes to thaw at room temperature for a maximum of 24 h on a
slowly rotating bed to ensure complete RBC lysis. Once thawed,
purifi cation begins with centrifugation to pellet the nucleic acids.
The pellet is washed and resuspended in buffer with proteinase K
to optimize protein digestion. The lysate is then centrifuged through
a Paxgene shredder spin column for homogeneity and to remove
cell debris. Then, through a process of washes and centrifugation,
RNA is eluted in buffer 5. There are multiple points in the protocol
that can be optimized to increase yield as indicated * throughout
the manufacturer procedures.

1. Centrifuge the PAXgene Blood RNA Tube for 10 min at
3,000-5,000× g using a swing-out rotor.
2. Remove the supernatant by decanting or pipetting. Add 4 ml
RNase-free water to the pellet, and close the tube using a fresh
secondary Hemogard closure. If the supernatant is decanted,
take care not to disturb the pellet, and dry the rim of the tube
with a clean paper towel. (*If you are experiencing low yield,
you can repeat this washing step.)
3. Vortex until the pellet is visibly dissolved, and centrifuge for
10 min at 3,000-5,000× g using a swing-out rotor. Remove
and discard the entire supernatant.
4. Add 350

Procedures

μ l buffer BR1 and vortex until the pellet is visibly

dissolved.
5. Pipet the sample into a 1.5 ml microcentrifuge tube. Add
300

l proteinase K. Mix by vortexing
for 5 s, and incubate for 10 min at 55°C using a shaker-incubator
set at maximum speed.
6. Pipet the lysate directly into a PAXgene Shredder spin column
(lilac) placed in a 2 ml processing tube, and centrifuge for
3 min at maximum speed (but not to exceed 20,000× g ).
7. Carefully transfer the entire supernatant of the fl ow-through
fraction to a fresh 1.5 ml microcentrifuge tube without dis-
turbing the pellet in the processing tube. (*Sometimes, the
pellet is viscous making it diffi cult to transfer the supernatant;
you can try another quick centrifuge to repellet.)
8. Add 350

μ l buffer BR2 and 40 μ

l ethanol (96-100%, purity grade p.a.) mix by vortex-
ing, and centrifuge briefl y (1-2 s at 500-1,000× g ) to remove drops
from the inside of the tube lid. (*If you are experiencing low yield,
you can repeat this step to increase RNA precipitation.)
9. Pipet 700

μ

l sample into the PAXgene RNA spin column (red)
placed in a 2 ml processing tube, and centrifuge for 1 min at

μ