Derivation: The following protocol is a modified Zhao Lai et al 2006 and TRizol Plus protocol.
Expected results: I tested the protocol with Arabidopsis seedlings and it gives quality RNA that was used for RT-PCR (non-quantitative). I am able to achieve 75-270 ng/uL (100 uL elution – take this value with a grain of salt since it is derived from the nanodrop) of RNA from 38-48 mg of seedlings (this is approximately 20 seedlings grown for 18 days). I have yet to experience a failed RT-PCR with RNA isolated using this extraction.
Keys to success: Yield is highly linked to tissue mass and quality of tissue grind. The range of tissue mass is very small. One should use a small tissue amount and process a given sample over a column many times. Tissue is ground successfully only when it is not visible in the trizol/tissue/bead mixture. Minimize RNAse paranoia – Guanidine isothiocyanate does a good job of destroying protein structure, inactivating RNAses.
More after the jump
****NOTE: Trizol is phenol based. Keep skin protected. Trizol will cause chemical burns. Keep glycerol on hand in case there is contact with skin.****
You will need the following:
Tissue lyser robot
Zirconia (2 mm) beads (x3-5)
Chrome (3 mm) beads (x3)
Trizol (fumehood, stored at RT) – 0.5 – 1.0 mL per sample
RNA free tubes (3x per sample)
RNAse away spray for gloves.
75-80% ethanol (made in RNAse free water)
100% ethanol Epoch DNA/RNA columns
Centrifuge at room temperature set to 12, 000 g
Use RNA free tubes. Prepare these on a surface sprayed with RNAse away or equivalent NaOH solution. Spray your tube rack with RNAase away.
1. Add 3 chrome beads and 3 zirconia beads to a tube.
2. Place tissue/seedlings in tube with beads
3. Add 500 uL of Trizol to tube containing tissue and beads. NOTE: Scale this to 1 mL Trizol for 100 mg.
4. Place tubes in tissue lyser for 3 minutes at 30 hz. With this treatment the tissue should become invisible. Keep grinding if you see chunks; you have time.
5. Pull tubes out of lyser and incubate tubes 5 min at RT.
6. Add 200 uL of chloroform to your macerated tissue/trizol mixture. Do not vortex. Vortexing will shear DNA and drive it into your RNA phase. Flick the tubes with your finger or invert vigorously to mix. The solution should become opaque.
7. Incubate 2 min at RT. This will allow your hydrophilic and hydrophobic phases to separate.
8. Centrifuge at room temperature: 10 min at 12,000 g
9 New tube: The top phase is aqueous. Transfer aqueous phase (should be clear to clear brown, if dark pink re-extract with chloroform).
10. Add 70-80% ethanol to this aqueous phase.
11. Invert gently until the solution is homogeneous.
12. Apply the solution from step 11 to a spin column.
13. Centrifuge for 30 sec (this can be as little as 15 sec but our centrifuge timer’s minimum is set to 30 sec
14. **DNAse treatment** If your sample must be DNA free for your downstream reaction/usage proceed to wash the column with 500 uL RW1.
If DNAse treatment is not necessary proceed to step 16. For each sample make a master mix of 10 uL DNAse and 70 RDD buffer (Both purchased from Qiagen). Apply DNAse diluted in buffer RDD to columns.
15. Incubate 15 minutes at room temperature. Add 500 uL of RW1 and spin out DNAses.
16. Add 700 uL 100% ethanol to spin column. Spin 30 sec, 12,000g
17. Discard flow through and wash column with 70-80% ethanol.
18. Repeat 17.
19. Decant flow through and spin the column for an additional 2 min to remove ethanol.
20. Place column in clean tube. Add 100 uL RNAse free water. Incubate 1-2 minutes.
21. Spin column at 12,000 g for 2 minutes