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

Author: Bin HE (hebin@uchicago.edu)

• 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 10 min (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) at 4-8C 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

[Optional Protocol for co-extraction of total DNA] -- borrowed from Misha Ludwig

1. To co-extract total DNA, save the bottom organic phase from step 5 above.
2. Add 100 μL 100% ethanol into the bottom phase, mix by inversion, store @RT for 10 min
3. Spin 2,000g for 5 min @4-8C to collect DNA pellet
4. Remove the phenol-ethanol supernatant.
5. Wash DNA pellet twice in a solution containing 0.1M sodium citrate in 10% ethanol. Use 300 ul for 300 ul of starting volume of TRIzol per vial. For each wash use 30min -45min @ RT (time to time mix by hands or just put on slow speed platform mixer). Before changing wash solution Spin 2,000 x g for 5 min at 4-8C.
6. Following these wash steps suspend DNA in 450-600 ul of 75% ethanol for 300 ul of TRIzol. Store 20 min at RT. Then Spin 2,000 x g for 5 min at 4-8C to get rid of 75% ethanol. Take out solution by pippetman aspiration.
7. Spin briefly (momentum) extra time to carefully take out a rest of solution in your sample vials.
8. Add to samples 10-20 μL of 8mM NaOH. The pH of the 8mM NaOH solution is ~9. Store samples at RT overnight.
9. Determine concentration on nanodrop, add EB Buffer (pH=8.6 from Qiagen) to each vial to get the desired concentration. Samples are ready for qPCR genotyping.