This work is licensed under CC BY-SA 4.0

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

  1. Collect 100~150 heads into a 1.5mL tube by liquid nitrogen flash freezing1.
  2. Add 150uL of cold TRIzol into the tube. Immediately, homogenize with motor-pestle.
  3. Add 850uL of TRIzol to a final 1mL volume. Mix well.
  4. Centrifuge lysate for 5min, 4C, full speed (>12k xg).
  5. Transfer the clear supernatant (>900uL) to a new tube. Avoid the debris pellet.
  6. Incubate at room temperature for 5min.
  7. Add 200uL of chloroform. Firmly cap the tube and shake rigorously for 20sec to mix2.
  8. Incubate at room temperature for 3min.
  9. Centrifuge 15min, 4C, full speed (>12k xg).
  10. Carefully transfer the upper aqueous phase (350~400uL) to a new tube. Do not disturb the white interphase nor the red lower phase.
  11. Add 500uL of isopropanol to the aqueous phase. Mix well by pipetting.
  12. Incubate on ice for 10min.
  13. Centrifuge 10min, 4C, full speed (>12k xg). Total RNA will be the white gel-like pellet.
  14. Discard supernatant. Add 1mL of freshly prepared 75% EtOH.
  15. Pipette to break the pellet to wash.
  16. Centrifuge 5min, 4C, 7.5k xg.
  17. 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.
  18. Discard all supernatant with P200 and P20 pipettes. Alternatively, pellet in EtOH may be store in -20C overnight.
  19. 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.
  20. Resuspend the pellet in 50uL ultrapure water with 0.1mM EDTA4 by pipetting.
  21. Incubate at 57C for 10min to fully dissolve the pellet. Vortex briefly then spin down.
  22. Measure absorbance with Nanodrop.
  23. 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 reagentVendor catalogStorage
TRIzol reagentInvitrogen 155964C
isopropanolSigma I9516Room temp in ventilated cabinet
chlorformSigma C2432Room temp in ventilated cabinet
100% ethanol (200 Proof)Fisher BP2818100Room temp in ventilated cabinet
0.5M EDTA pH8.0, RNase-freeInvitrogen AM9260GRoom temp
ultrapure waterInvitrogen 10977Room temp

  1. Importantly, the fly heads should remain frozen during this step to prevent degradation. After heads are put in TRIzol, they shall be homogenized immediately. ↩︎

  2. Rigorous shaking is important to create a thorough emulsion and increase RNA yield. However, it will shear genomic DNA and increase DNA contamination. ↩︎

  3. Breaking the pellet is important to eliminate salts in the TRIzol, most notably guanidinium thiocyanate (GITC). ↩︎

  4. EDTA is important to prevent RNA hydrolysis by divalent ions. However, excessive EDTA may inhibit enzymes in downstream applications. ↩︎

  5. TRIzol RNA extraction will inevitably introduce DNA contamination that may be significant (>10%) . DNase treatment is therefore required for many downstream applications. ↩︎

  6. 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. ↩︎