Characterization of RNA - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 132

8,000-20,000× g . Place the spin column in a new 2 ml
processing tube, and discard the old processing tube contain-
ing fl ow-through.
10. Pipet the remaining sample into the PAXgene RNA spin col-
umn, and centrifuge for 1 min at 8,000-20,000× g . Place the
spin column in a new 2 ml processing tube, and discard the old
processing tube containing fl ow-through.
11. Pipet 350 l buffer BR3 into the PAXgene RNA spin column.

Centrifuge for 1 min at 8,000-20,000× g . Place the spin
column in a new 2 ml processing tube, and discard the old
processing tube containing fl ow-through.
12. Add 10

μ

l buffer RDD in a
1.5 ml microcentrifuge tube and mix by gently fl icking the
tube, and centrifuge briefl y to collect residual liquid from the
sides of the tube.
13. Pipet the DNase I incubation mix (80

μ l DNase I stock solution to 70 μ

l) directly onto the
PAXgene RNA spin column membrane, and place on the
benchtop (20-30°C) for 15 min.
14. Pipet 350

μ

l buffer BR3 into the PAXgene RNA spin column,
and centrifuge for 1 min at 8,000-20,000× g . Place the spin
column in a new 2 ml processing tube, and discard the old
processing tube containing fl ow-through.
15. Pipet 500

μ

l buffer BR4 to the PAXgene RNA spin column,
and centrifuge for 1 min at 8,000-20,000× g . Place the spin
column in a new 2 ml processing tube, and discard the old
processing tube containing fl ow-through.
16. Add another 500

μ

l buffer BR4 to the PAXgene RNA spin
column. Centrifuge for 3 min at 8,000-20,000× g .
17. Discard the tube containing the fl ow-through, and place the
PAXgene RNA spin column in a new 2 ml processing tube.
Centrifuge for 1 min at 8,000-20,000× g .
18. Discard the tube containing the fl ow-through. Place the
PAXgene RNA spin column in a 1.5 ml microcentrifuge tube,
and pipette 40

μ

l buffer BR5 directly onto the PAXgene RNA
spin column membrane. Centrifuge for 1 min at 8,000-
20,000× g to elute the RNA.
19. Repeat the elution step (step 18) as described, using 40

μ

l buffer
BR5 and the same microcentrifuge tube. (*If you are experienc-
ing low yield, try eluting in a total of 40

μ

l.)
20. Incubate the eluate for 5 min at 65°C in the shaker-incubator
(from step 5) without shaking. After incubation, chill RNA
immediately on ice.
21. If the RNA samples are not used immediately, store at −20
or −80°C. Since the RNA remains denatured after repeated

μ l (20 and 20 μ