RNA prep from CRC with DNase I treatment
Date: 10/28/20
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!
RNasin: Recombinant RNasin® Ribonuclease Inhibitor (Promega, Cat # N2515)
RNasin will be used at varying concentrations; Prepare individual tubes of each buffer for each sample
Direct-zol RNA MicroPrep (Zymo Research, Cat # R2060)
Extraction/Wash buffers with Protease and RNase inhibitors: 20mM HEPES, pH7.4, 500mM NaCl, 1% Triton X-100 with or without 25mM EDTA
Extraction/Wash buffers were made with Nuclease-free water (the stock solutions are nuclease free) with Protease inhibitors used at 1x during extraction and washes; RNasin was used @ 1:40 in extraction buffer and 1:250 in wash buffer
Cell Line / CRC:
197 Adenocarcinoma (Colon, T/N)
268 Ovarian carcinoma (Ovary, T/N)
174 Metastatic carcinoma (Breast, T)
Scale: 50mg scale with 10ul anti-ORF1 beads per reaction
Weigh out the following powder
2x 50mg of 197T (+/- EDTA)
2x 50mg of 197N (+/- EDTA)
2x 50mg of 268T (+/- EDTA)
2x 50mg of 268N (+/- EDTA)
2x 50mg of 174T (+/-EDTA)
Resuspend the powder by vortexing (2, 3, 5, 7 and 9; see below IP set up for numbering )
Resuspend the powder by vortexing plus sonication (1, 4, 6, 8 and 10; see below IP set up for numbering )
To shorten the time for sonication, I used the old sonicator, 1 sec; setting 3
197N and 268N cell extract was very viscous, sonication reduced viscosity for most of the samples except 268N
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Save 20ul of tumor supernatant for RNA prep (“Input”); snap freeze the input directly. Since there was a problem with the sample extraction, input samples may not be used for RNA prep.
Lysate from 268N was very viscous. <100ul of clarified cell extract was recovered from #8 268N in EB with EDTA with sonication; left a huge "pellet" behind. Picture below shows pellet left in each tube from left to right 1-10 (same order as IP setting)
During spin, wash 60ul anti-ORF1 beads with extraction buffer that does NOT have RNasin added (to conserve RNasin).
Set up IP with 10ul of anti-ORF1 beads per 50mg reaction
1) 197T-EDTA
2) 197T+EDTA
3) 197N-EDTA
4) 197N+EDTA
5) 268T-EDTA
6) 268T+EDTA
7) 268N-EDTA
8) 268N+EDTA
9) 174T-EDTA
10) 174T+EDTA
IP @ 4°C for15’ (get tubes and Zymo columns ready while IP is going)
Save 20ul of supernatant (“FT”); snap freeze RNA portion in 250ul Trizol in liquid nitrogen (Skip RNA prep for FT this time)
Wash 2x 250ul with wash buffer
Switch beads to fresh tubes at 2nd wash step Trizol Elution: Elute in 250ul of Trizol
RNA extraction (Keep input and FT in Trizol and stored @ -80C; precess elution samples)
- all steps from here may be performed at RT if not mentioned otherwise
Vortex for 1min, place the tubes aside (can be on ice or RT, Trizol extract is stable at RT) Spin the PhsMkr tube at 16k RCF, 30 sec
Add 50ul ChlrFm and 25ul water to the PhsMkr do this immediately before adding TriZol elutions or (perhaps safer still) immediately after transfering elutions to the PhsMkr tube. Adding ChlrFm/water too far in advance may cause a failure. not adding the water will cause the PhsMkrto fail and 'swallow' the aqueous portion of the TriZolreagent mix.
Collect the elution (“E”, pulse spin, place on magnet) and transfer to the PhsMkr tube
Mix by hand, vigorously for 15 sec
Incubate 2 min @ RT with end-over-end mixing this step is almost certainly dispensable for IP - it is supposed to be time for RNPs to dissociate.
Spin 16k RCF, 4C (not important, Trizol extract is stable at RT), 5 min
Transfer the aqueous phase to a new tube by pressing pipette tip against wall of tube; avoid puncturing the Phasemaker gel
Add an equal volume of 100% EtOH (~185uL) 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 @ 16,000rcf for 30 seconds
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 (20-30C) 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 @ 16,000rcf 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.
Send 1.5ul of Elution for pico chip bioanalyzer analysis.
Bioanalyzer result (Elution 1-10, see IP set up)
https://drive.google.com/file/d/1j2_kwhKwYUZZ8kfluubnb75xfy6c3at4/view?usp=sharing
Conclusion:
DNase I treatment helped clean up the total RNA prep
Need to improve the extraction condition for normal tissue especially in the presence of EDTA