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

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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''
* 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===
===Protocol===
===Protocol===
# Thaw sample (in TRIzol, -80C) @RT, stay for 10min (to ensure that samples are completely dissolved by TRIzol
# Thaw sample (in TRIzol, -80C) @RT, '''stay for 10 min''' (to ensure that samples are completely dissolved by TRIzol
# For 300ul TRIzol, add 60ul <math>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.
# For 300μL TRIzol, add 60μL <math>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'''.
# Spin 12,000g (I use max speed on a desktop eppendorf centrifuge, at 13,600rpm) for 15 min.
# Spin '''12,000g''' (I use max speed on a desktop eppendorf centrifuge, at 13,600rpm) '''at 4-8C for 15 min'''.
# Prepare 3 tubes with 80ul of <math>CHCl_{3}</math> while centrifuging.
# While centrifuging, prepare new tubes with '''60μL of <math>CHCl_{3}</math>'''.
# After centrifugation, carefully transfer the top aqueous phase to the new tube with <math>CHCl_{3}</math> from the last step.  
# After centrifugation, '''carefully*''' transfer the top aqueous phase (safe to take '''130μL''') to the new tube with <math>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
# Shake vigorously for 15 sec, let sit for 2min, spin @4C for 15 min
          but the interphase still contains proteins and perhaps some organic phase. Usually from 300ul TRIzol, I take about ? ul.
# While centrifuging, prepare new tubes with '''100 μL of 70% Ethanol'''. (prepared with RNase free water)
# This time carefully transfer about 100 μL of the top aqueous phase to a new tube.
# 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).
 
<nowiki>*</nowiki>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
# To co-extract total DNA, save the bottom organic phase from step 5 above.
# Add 100 μL 100% ethanol into the bottom phase, mix by inversion, store @RT for 10 min
# Spin '''2,000g for 5 min @4-8C''' to collect DNA pellet
# Remove the phenol-ethanol supernatant.
# 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'''.
# 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.
# Spin briefly (momentum) extra time to carefully take out a rest of solution in your sample vials.
# Add to samples 10-20 μL of 8mM NaOH.  The pH of  the 8mM NaOH solution is ~9. Store samples at RT overnight.
# 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.

Latest revision as of 12:32, 9 April 2012

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

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 [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.
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