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 ERDC TN-DOER-R4
September 2004
RNA Isolation
Procedure
Transfer 40 x 106 cells to a sterile 50-mL conical tube.
1.
Centrifuge at 500 x g for 5 minutes at 4EC; discard supernatant.
2.
3.
Rinse pellet with ~25 mL of 1X PBS buffer (rinse cell pellet in RNA Later the same way).
Centrifuge at 500 x g for 5 minutes at 4EC; discard supernatant. Repeat rinse.
4.
5.
Add 1.0 ml denaturing solution, and IMMEDIATELY vortex to mix.
6.
Pass contents through a syringe with a 26 gauge needle ~10 times to shear DNA or until the
DNA strands are no longer lumpy. Transfer contents into a 1.7-mL microcentrifuge tube.
7.
Allow resuspended pellet to sit on ice for 5 - 10 minutes.
8.
Vortex, then centrifuge at 15,000 x g for 5 minutes at 4C.
9.
Transfer supernatant into two new microcentrifuge tubes.
10.
Add 0.67 mL of saturated phenol to each tube and vortex for ~1 minute.
11.
Incubate the tubes on ice for 5 minutes.
12.
Add 0.2 mL of chloroform to each tube, shake and vortex for 1 - 2 minutes.
13.
Incubate the tubes on ice for 5 minutes.
14.
Centrifuge at 15,000 x g for 10 minutes at 4C.
15.
Transfer top aqueous phase to a new tube, repeat steps 10 - 14 another three times for a total
of four Phenol-Chloroform extractions. Avoid pipetting into the interphase or organic phase.
16.
Transfer top aqueous phase to a new tube and add 0.67 mL isopropanol slowly.
17.
Mix well and let tubes sit on ice for 10 minutes.
18.
Centrifuge at 15,000 x g for 10 minutes at 4C.
19.
Carefully remove and discard supernatant. Use care not to disturb the RNA pellet.
20.
Add 0.5 mL of 80 percent ethanol.
21.
Centrifuge at 15,000 x g for 5 minutes at 4C.
22.
Carefully remove and discard supernatant. Use care not to disturb the RNA pellet.
23.
Air dry pellet.
24.
Resuspend RNA pellet in RNase-free water to about 2 g/L (about 20 L/tube) and
combine RNA from the same treatment into a tube.
25.
Determine RNA purity and yield [see details next page].
Perform phenol:chloroform cleanup if the RNA sample is still contaminated with proteins.
26.
27.
Continue with DNase treatment of RNA, or store RNA at -80C for treatment later.
DNase Treatment of RNA
Materials needed
RNA sample
1.7-mL microcentrifuge tubes (Rnase-DNase free)
10X DNase buffer (kit)
Air incubator (37C)
DNase 1 (kit)
Tabletop centrifuge
RNase-free water (kit)
10-L pipet and pipet tips
10X termination mix (kit)
100-L pipet and pipet tips
Chloroform (Sigma #C-2432)
96 percent ethanol (Aldrich #E702-3)
2M NaOAc (kit)
80 percent ethanol (Aldrich #E702-3)
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