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Process
Avoid RNase contamination throughout the process. For example, wear gloves and lab coat all the time, use filtered RNase-free pipette tips, and keep bench surfaces clean.
Typical yield of this protocol is ~10ug total RNA from ~100 fly heads with pure RNA Nanodrop metrics (A260/280 ~2.0, A260/230 ~2.1).
Formal citation of this protocol will be available at a later time (expected: August 2024).
TRIzol extraction
- Collect 100~150 heads into a 1.5mL tube by liquid nitrogen flash freezing1.
- Add 150uL of cold TRIzol into the tube. Immediately, homogenize with motor-pestle.
- Add 850uL of TRIzol to a final 1mL volume. Mix well.
- Centrifuge lysate for 5min, 4C, full speed (>12k xg).
- Transfer the clear supernatant (>900uL) to a new tube. Avoid the debris pellet.
- Incubate at room temperature for 5min.
- Add 200uL of chloroform. Firmly cap the tube and shake rigorously for 20sec to mix2.
- Incubate at room temperature for 3min.
- Centrifuge 15min, 4C, full speed (>12k xg).
- Carefully transfer the upper aqueous phase (350~400uL) to a new tube. Do not disturb the white interphase nor the red lower phase.
- Add 500uL of isopropanol to the aqueous phase. Mix well by pipetting.
- Incubate on ice for 10min.
- Centrifuge 10min, 4C, full speed (>12k xg). Total RNA will be the white gel-like pellet.
- Discard supernatant. Add 1mL of freshly prepared 75% EtOH.
- Pipette to break the pellet to wash.
- Centrifuge 5min, 4C, 7.5k xg.
- Discard supernatant. Repeat the EtOH wash two times for a total for three washes. Subsequent washes: add 100uL 75% EtOH, vortex briefly to break pellet3, fill to 1mL.
- Discard all supernatant with P200 and P20 pipettes. Alternatively, pellet in EtOH may be store in -20C overnight.
- Air dry the RNA pellet until no EtOH remains (5-10min). Do not let the pellet dry out completely. Otherwise the RNA will be hard to redissolve.
- Resuspend the pellet in 50uL ultrapure water with 0.1mM EDTA4 by pipetting.
- Incubate at 57C for 10min to fully dissolve the pellet. Vortex briefly then spin down.
- Measure absorbance with Nanodrop.
- Proceed to DNase treatment immediately5.
DNase treatment
Various options exist for DNase treatment, most notably on-column and in-solution variants. For example, one option I use in the lab is TURBO DNA-free kit. Follow manufacturerc protocol to perform DNase treatment.
After treatment, measure final RNA concentration again with Nanodrop and/or Qubit6.
RNA may be used immediately or stored as aliquots (15~20uL each) in -80C. Avoid repeated freeze-thaw cycles.
Material and Storage
Stock reagent | Vendor catalog | Storage |
---|---|---|
TRIzol reagent | Invitrogen 15596 | 4C |
isopropanol | Sigma I9516 | Room temp in ventilated cabinet |
chlorform | Sigma C2432 | Room temp in ventilated cabinet |
100% ethanol (200 Proof) | Fisher BP2818100 | Room temp in ventilated cabinet |
0.5M EDTA pH8.0, RNase-free | Invitrogen AM9260G | Room temp |
ultrapure water | Invitrogen 10977 | Room temp |
Importantly, the fly heads should remain frozen during this step to prevent degradation. After heads are put in TRIzol, they shall be homogenized immediately. ↩︎
Rigorous shaking is important to create a thorough emulsion and increase RNA yield. However, it will shear genomic DNA and increase DNA contamination. ↩︎
Breaking the pellet is important to eliminate salts in the TRIzol, most notably guanidinium thiocyanate (GITC). ↩︎
EDTA is important to prevent RNA hydrolysis by divalent ions. However, excessive EDTA may inhibit enzymes in downstream applications. ↩︎
TRIzol RNA extraction will inevitably introduce DNA contamination that may be significant (>10%) . DNase treatment is therefore required for many downstream applications. ↩︎
In-solution DNase treatment like TURBO can introduce salts that interfere with Nanodrop reading. Therefore, you cannot get reliable contaminant analysis from Nanodrop at this stage. For a more accurate RNA concentration measurement, fluorometers such as Qubit or Quantus are recommended. ↩︎