Total RNA prep using Direct-zol RNA MicroPrep kit with on column DNase I treatment
Reference RNA extraction after cell powder IP. This is a very similar protocol without DNase I treatment.
Everything should be done RNA-grade with nuclease-free reagents, clean bench and pipets with RNase Zap; Use newly prepared buffers and new boxes of tips and tubes etc. whenever reasonably possible. Change gloves often!
Reagents
RNasin: Recombinant RNasin® Ribonuclease Inhibitor (Promega, Cat # N2515)
cOmplete™, EDTA-free Protease Inhibitor Cocktail (Sigma, Cat# 11873580001)
TRI Reagent® (ThermoFisher Scientific, Cat # 15596026)
Phasemaker tubes (ThermoFisher Scientific, Cat # A33248)
Direct-zol RNA MicroPrep (Zymo Research, Cat # R2060)
Other RNA grade reagents: chloroform, ethanol, nuclease free water
Protocol
I. Lyse and homogenize samples in Trizol according to your starting material
We normally start with 100mg of cryo-milled cell or tissue powder
Add 4 vol. of extraction buffer with protease inhibitors and RNasin to powder 1:4 w:v (e.g. 400ul of extraction buffer to 100mg of powder)
One of our typical extraction buffer is 20mM HEPES, pH7.4, 500mM NaCl, 1% Triton X-100
Resuspend the powder by vortexing, can add a brief sonication step if powder is hard to resuspend
Spin @ 20,000 rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Save 5 to 10% of supernatant for RNA prep and use the rest for affinity purification of target protein
Add 250ul of Trizol to the fraction saved for RNA prep If you don’t isolate RNA right after, snap freeze the sample in liquid N2 and stored at -80°C
II. Phase separation
Vortex for 1 minute, place the tubes aside (can be on ice or RT, Trizol extract is stable at RT)
Spin the Phasemaker tube at 16,000 rcf, 30 seconds
Add 50ul chloroform and 25ul water to the Phasemaker, do this immediately before adding your sample in Trizol or (perhaps safer still) immediately after transfering sample to the Phasemaker tube. Adding chloroform/water too far in advance may cause a failure. not adding the water will cause the Phasemaker to fail and 'swallow' the aqueous portion of the Trizol reagent mix.
Add sample (in 250ul of Trizol) to the Phasemaker tube
Mix by hand, vigorously for 15 seconds
Incubate 2 minutes @ RT with end-over-end mixing - it is supposed to be time for RNPs to dissociate.
Spin 16,000 rcf, 4°C (not important, Trizol extract is stable at RT), 5 minutes
Transfer the aqueous phase to a new tube by pressing pipette tip against wall of tube; avoid puncturing the Phasemaker gel
III. RNA precipitation and purification using Direct-zol RNA MicroPrep kit https://fnkprddata.blob.core.windows.net/domestic/data/datasheet/ZYR/R2060.pdf
Add an equal volume of 100% EtOH to each sample and mix thoroughly, vortex and pulse spin
Transfer the mixture to a Zymo-Spin IC column in a collection tube and centrifuge @ 16,000 rcf for 30 seconds (to process samples > 700ul, reload the column and repeat Step 2)
DNase I treatment
• Add 400ul RNA Wash Buffer to the column and centrifuge @ 16,000rcf for 30 sec
• In an RNase-free tube, add 5ul DNase I 95U/ul), 35ul DNA Digestion Buffer and mix. Add the mix directly to the column
• Incubate @ RT for 15 minutes
Add 400ul Direct-zol RNA PreWash to the column and centrifuge @ 16,000rcf for 30 seconds. Discard the FT and repeat this step. Use the vacuum-trap system to clear the liquid from the tubes both times
Add 700ul RNA Wash Buffer to the column and centrifuge for 2 minutes @ 16,000 rcf to ensure complete removal of the wash buffer. Transfer the column carefully into an RNase-free tube.
To elute RNA, add 6ul of RNase-Free Water directly to the column matrix and centrifuge @ 16,000rcf for 30 seconds.
QC of RNA samples by Bioanalyzer: Send ~1ul of Elutions for pico chip bioanalyzer analysis.
Saved the remainder for RNA-seq analysis or other downstream applications. Store remainder RNA at -80°C.