RNA prep from CRC with hUdT
Date: 04/19/21
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)
hUdT: 3'-N-hydroxyurea-3'- deoxythymidine 5'-monophosphate, in free form (from Ali Alipour Najmi Iranag, University of Groningen). MW: 380.25g/mole (1M->380.25g/L)
Direct-zol RNA MicroPrep (Zymo Research, Cat # R2060)
Making hUdT stock
Started by making 100mM stock of hUdT: dissolve 38mg compound in 100ul MeOH. We have 5.2mg @95% purity; should be dissolved in 130ul (4.94mg/38mg/ml=0.13ml or130ul). The compound did not dissolve well, there was a big pellet after brief spin
Add antother 130ul of MeOH, the concentration decreased to 50mM, still did not dissolve; total volume increased to 260ul.
Add water to the solution, 10ul of water at a time. After adding 50ul of water, the solution finally became clear. Final volume was 310ul, final concentration was 42mM. Will use this as 40mM stock (200x).
Extraction/Wash buffers with Protease and RNase inhibitors:
20mM HEPES, pH7.4, 500mM NaCl, 1% Triton X-100 with 1mM ZnCl2 20mM Tris, pH8, 500mM NaCl, 1% Triton X-100 with 1mM ZnCl2
I tested NaPhosphate (NaP) and Tris buffer. Starting with 0.5M NaPhosphate pH8 and 1M Tris, pH8. Making 20 and 40mM buffer with 500mM NaCl and 1% Triton.
20mM NaP buffer, pH was 7.4 before adding to powder; the pH of 1:4 w:v clarified lysate was 6.8.
40mM NaP buffer, pH was 7.9 before adding to powder; the pH of 1:4 w:v clarified lysate was 7.4.
I added more NaP stock solution to the lysate. When final NaPhosphate reached 100mM, pH of the lysate went back to 8.
This suggests that NaPhosphate buffer is not good at maintaining pH. If we want to use it as extraction buffer, we need to use it @ 100mM.
Tris buffer is good. Cell lysate maintained pH 8 using both 20mM of 40mM Tris buffer @ 1:4 w:v.
Based on the test above, we will use 20mM Tris buffer when testing RNase inhibitor at pH8.
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 with or without hUdT @ 200um and 1:250 in wash buffer
CRC tissues 162 Metastatic sigmoid colon cancer (Liver, T; previously failed to get RNA from elution) 174 Metastatic carcinoma (Breast, T; positive control; previously got RNA from elution)
Scale: 50mg scale with 10ul anti-ORF1 beads per reaction
Weigh out 4x 50mg of 162T, 4x 50mg of 174T
Add 200ul of extraction buffer with protease inhibitors and RNase inhibitors to each tube according to the table below
Resuspend the powder by vortexing
Sonicate 1 sec @ setting 3
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Save 20ul of tumor supernatant for RNA prep (“Input”); Add 250ul of Trizol and snap freeze
During spin, wash 80ul anti-ORF1 beads with extraction buffer
Set up IP with 10ul of anti-ORF1 beads per 50mg reaction
IP @ 4°C for 15’ (get tubes and Zymo columns ready while IP is going)
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 Phasemaker tube at 16k RCF, 30 sec
Add 50ul of chloroform and 25ul H2O to the Phasemaker tube do this immediately before adding elutions or (perhaps safer still) immediately after transferring elutions to the Phasemaker tube. Adding chloroform / H2O too far in advance may cause a failure. not adding the water will cause the Phasemaker tube to fail and 'swallow' the aqueous portion of the Trizol reagent mix.
Collect the elution (“E”, pulse spin, place on magnet) and transfer to the Phasemaker 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. (11 samples on 1 chip; omit input samples of 174T and 1 input of 162T; save those samples in -80C freezer)
1) Input_162T_HEPES, pH7.4_RNasin
2) Input_162T_HEPES, pH7.4_RNasin+ hUdT
3) Input_162T_Tris, pH8_RNasin+ hUdT
4) E_162T_HEPES, pH7.4_RNasin
5) E_162T_HEPES, pH7.4_RNasin+ hUdT
6) E_162T_Tris, pH8_RNasin
7) E_162T_Tris, pH8_RNasin+ hUdT
8) E_174T_HEPES, pH7.4_RNasin
9) E_174T_HEPES, pH7.4_RNasin+ hUdT
10) E_174T_Tris, pH8_RNasin
11) E_174T_Tris, pH8_RNasin+ hUdT
Bioanalyzer result
https://drive.google.com/file/d/1BVC_KWsY4oQnteFSTYjErbglyY_N2upx/view?usp=sharing