Kreitman:RNA extraction from small amount of samples (imaginal discs)

• This protocol is optimized for extracting total RNA using TRIzol + column method from small amount of samples. In my case I used eye imaginal discs dissected from 10 third instar wandering larvae of Drosophila melanogaster

Protocol

1. Thaw sample (in TRIzol, -80C) @RT, stay for 10min (to ensure that samples are completely dissolved by TRIzol
2. For 300μL TRIzol, add 60μL [math]\displaystyle{ CHCl_{3} }[/math] (Use 100μL for 500μL of TRIzol, similar for the rest of the protocol). Shake vigorously for 15 sec, let sit for 2 min @RT.
3. Spin 12,000g (I use max speed on a desktop eppendorf centrifuge, at 13,600rpm) @4C for 15 min.
4. While centrifuging, prepare new tubes with 60μL of [math]\displaystyle{ CHCl_{3} }[/math].
5. After centrifugation, carefully* transfer the top aqueous phase (safe to take 130μL) to the new tube with [math]\displaystyle{ CHCl_{3} }[/math] from the last step.
6. Shake vigorously for 15 sec, let sit for 2min, spin @4C for 15 min
7. While centrifuging, prepare new tubes with 100 μL of 70% Ethanol. (prepared with RNase free water)
8. This time carefully transfer about 100 μL of the top aqueous phase to a new tube.
9. Vortex for 15 sec, transfer to RNeasy column, continue with the RNeasy protocol (there is a special protocol to be used in conjunction with a TRIzol based extraction method).

*Be careful not to take any of the interphase. For this small amount of samples, you are not going to see a cloudy interphase. But the interphase still contains proteins and perhaps some organic phase. For 500μL TRIzol + 100μL CHCl3, take 200~220 μL; for 300 + 60, take ~130 μL