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

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Author: Bin HE
* 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''
* 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''
===Reagents===
===Reagents===

Revision as of 15:31, 9 April 2012

Author: Bin HE

  • 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

Reagents

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 CHCl3 (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 CHCl3.
  5. After centrifugation, carefully* transfer the top aqueous phase (safe to take 130μL) to the new tube with CHCl3 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.
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