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 300ul TRIzol, add 60ul [math]\displaystyle{ CHCl_{3} }[/math] (Use 100ul for 500ul 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) for 15 min.
4. Prepare 3 tubes with 80ul of [math]\displaystyle{ CHCl_{3} }[/math] while centrifuging.
5. After centrifugation, carefully transfer the top aqueous phase to the new tube with [math]\displaystyle{ CHCl_{3} }[/math] from the last step.

Note: 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. Usually from 300ul TRIzol, I take about ? ul.