Trizol extraction for RNA and DNA

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This is a protocol for harvesting both RNA and DNA from mammalian tissue.  It is currently written for tissue preparation but can also be used on cell suspensions and cells grown on a monolayer.
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This is a protocol for extracting both RNA and DNA from mammalian tissue.  It is currently written for tissue preparation but can also be used on cell suspensions and cells grown on a monolayer. See here for an [[RNA extraction|overview of RNA extraction methods]].
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==Materials==
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*TRIzol or TRIzol LS reagent from [https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=189 Invitrogen]
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:*TRIzol LS is specifically created to work with liquid samples (i.e. blood and cell culture) it should be diluted when used with tissue samples.  The equivalent of 1ml of regular TRIzol is 250ul of H<sub>2</sub>O and 750ul of TRIzol LS.
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*0.1M Sodium Citrate in 10% Ethanol (for 100ml 2.94g of the sodium citrate dihydrate mixed with 10ml EtOH and brought up to 100ml with H<sub>2</sub>O)
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*100% Ethanol
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*75% Ethanol
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*8mM NaOH
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*TNES-6U (10mM Tris-HCl, pH 7.5; 125mM NaCl; 10mM EDTA pH 8.0; 1% SDS; 6M Urea) Note: The urea will not completely dissolve in solution (ie cloudy) so pre-heat to 40C prior to use.
==RNA==
==RNA==
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*Homogonize tissue in 1ml trizol per 50-100 mg of tissue without having more than 10% sample volume compared to trizol
*Homogonize tissue in 1ml trizol per 50-100 mg of tissue without having more than 10% sample volume compared to trizol
*Incubate sample 5' @ RT
*Incubate sample 5' @ RT
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===Extract RNA===
===Extract RNA===
*Add 0.2ml of chloroform per 1mL of trizol and shake for 15"
*Add 0.2ml of chloroform per 1mL of trizol and shake for 15"
*Spin at 12,000xg for 15' @ 4C
*Spin at 12,000xg for 15' @ 4C
*Remove top aqueous phase (SAVE bottom organic phase for [[Trizol extraction for RNA and DNA#DNA|DNA]])
*Remove top aqueous phase (SAVE bottom organic phase for [[Trizol extraction for RNA and DNA#DNA|DNA]])
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===RNA Precipitation===
===RNA Precipitation===
*Add 0.5ml of isopropanol per 1ml of trizol to the aqueous phase
*Add 0.5ml of isopropanol per 1ml of trizol to the aqueous phase
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*Wash pellet  with 1ml 75% EtOH per 1ml  trizol then vortex
*Wash pellet  with 1ml 75% EtOH per 1ml  trizol then vortex
*Spin at 12,000xg for 5' @ 4C
*Spin at 12,000xg for 5' @ 4C
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===RNA Resuspension===
===RNA Resuspension===
*Air dry for 5-10' and resuspend in 30-50ul RNAse free H2O
*Air dry for 5-10' and resuspend in 30-50ul RNAse free H2O
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==DNA==
==DNA==
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===Percipitation of DNA===
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===Precipitation of DNA (Ethanol)===
*Take organic phase and carefully remove all aqueous phase
*Take organic phase and carefully remove all aqueous phase
*Add 0.4ml 100% EtOH per 1ml of trizol mix by hand
*Add 0.4ml 100% EtOH per 1ml of trizol mix by hand
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*Spin at 2,000xg for 5' @ 4C
*Spin at 2,000xg for 5' @ 4C
*Remove sup (can be saved for protein purification)
*Remove sup (can be saved for protein purification)
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===DNA Wash===
===DNA Wash===
*Wash pellet 2x with 0.1M Sodium Citrate 10% EtOH 1ml per 1ml of trizol
*Wash pellet 2x with 0.1M Sodium Citrate 10% EtOH 1ml per 1ml of trizol
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*Resuspend in 1.5-2.0ml 75% EtOH and incubate for 10-20' @ RT mix periodically
*Resuspend in 1.5-2.0ml 75% EtOH and incubate for 10-20' @ RT mix periodically
*Spin at 2,000xg for 5' @ 4C
*Spin at 2,000xg for 5' @ 4C
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===Redissolve DNA===
===Redissolve DNA===
*Redissolve pellet in 300-600ul of 8mM NaOH per 50-70mg of tissue
*Redissolve pellet in 300-600ul of 8mM NaOH per 50-70mg of tissue
*Spin at 12,000xg for 10 minutes to remove cell detritus
*Spin at 12,000xg for 10 minutes to remove cell detritus
*Save supernatant with DNA
*Save supernatant with DNA
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===Precipitation of DNA (Back extraction)===
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This is an alternative protocol to the Ethanol precipitation protocol and begins
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at the same point; with the organic phase and the interphase after collecting the
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aqueous phase.
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*Add 300uL of 40C TNES-6U back extraction buffer per 1mL of Trizol to the organic
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phase and interphase
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*Shake by hand to mix then incubate at room temperature for 10 min.
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*Spin at 18,000xg for 15min at 4C in a fixed angle table top centrifuge
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*Transfer the aqueous phase to a clean tube (DNA) and discard the interphase (Protein)
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and the organic phase
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*Add an equal volume of isopropyl alchohol to the aqueous phase then incubate at -80C for 2hr
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*Spin at 18,000xg for 15min at 4C then discard the supernatant
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===DNA Wash===
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*Wash the pellet 3 times with 70% ethanol (incubate 3min, spin 18,000xg for 5min)
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*Air dry on bench for 5-10min or until liquid is not visible
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===Redissolve DNA===
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*Redissolve as described above, or in 300-500uL of 65C Low EDTA TE buffer pH 8.0 (Affymetrix)
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==Notes==
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*The back extraction buffer can increase the yields of DNA recovery by a significant amount compared to the described Ethanol protocol above.  I have tried both in the isolation of RNA and DNA from rhesus skin biopsies and the difference was significant.
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*The TNES-6U buffer protocol was adopted from the following paper:[http://www.ncbi.nlm.nih.gov/pubmed?term=18840898 "Simultaneous Extraction of High-Quality RNA and DNA from Small Tissue Samples".]
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==See also==
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* [[RNA extraction]]
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[[Category:Protocol]]
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[[Category:RNA]]

Current revision

This is a protocol for extracting both RNA and DNA from mammalian tissue. It is currently written for tissue preparation but can also be used on cell suspensions and cells grown on a monolayer. See here for an overview of RNA extraction methods.

Contents

Materials

  • TRIzol LS is specifically created to work with liquid samples (i.e. blood and cell culture) it should be diluted when used with tissue samples. The equivalent of 1ml of regular TRIzol is 250ul of H2O and 750ul of TRIzol LS.
  • 0.1M Sodium Citrate in 10% Ethanol (for 100ml 2.94g of the sodium citrate dihydrate mixed with 10ml EtOH and brought up to 100ml with H2O)
  • 100% Ethanol
  • 75% Ethanol
  • 8mM NaOH
  • TNES-6U (10mM Tris-HCl, pH 7.5; 125mM NaCl; 10mM EDTA pH 8.0; 1% SDS; 6M Urea) Note: The urea will not completely dissolve in solution (ie cloudy) so pre-heat to 40C prior to use.

RNA

Homoginize sample

  • Homogonize tissue in 1ml trizol per 50-100 mg of tissue without having more than 10% sample volume compared to trizol
  • Incubate sample 5' @ RT

Extract RNA

  • Add 0.2ml of chloroform per 1mL of trizol and shake for 15"
  • Spin at 12,000xg for 15' @ 4C
  • Remove top aqueous phase (SAVE bottom organic phase for DNA)

RNA Precipitation

  • Add 0.5ml of isopropanol per 1ml of trizol to the aqueous phase
  • Incubate 10' @ RT
  • Spin at 12,000xg for 10' @ 4C
  • Wash pellet with 1ml 75% EtOH per 1ml trizol then vortex
  • Spin at 12,000xg for 5' @ 4C

RNA Resuspension

  • Air dry for 5-10' and resuspend in 30-50ul RNAse free H2O

DNA

Precipitation of DNA (Ethanol)

  • Take organic phase and carefully remove all aqueous phase
  • Add 0.4ml 100% EtOH per 1ml of trizol mix by hand
  • Incubate 2-3' @ RT
  • Spin at 2,000xg for 5' @ 4C
  • Remove sup (can be saved for protein purification)

DNA Wash

  • Wash pellet 2x with 0.1M Sodium Citrate 10% EtOH 1ml per 1ml of trizol
  • At each wash incubate in wash solution for 30' @ RT
  • Spin at 2,000xg for 5' @ 4C
  • Resuspend in 1.5-2.0ml 75% EtOH and incubate for 10-20' @ RT mix periodically
  • Spin at 2,000xg for 5' @ 4C

Redissolve DNA

  • Redissolve pellet in 300-600ul of 8mM NaOH per 50-70mg of tissue
  • Spin at 12,000xg for 10 minutes to remove cell detritus
  • Save supernatant with DNA

Precipitation of DNA (Back extraction)

This is an alternative protocol to the Ethanol precipitation protocol and begins at the same point; with the organic phase and the interphase after collecting the aqueous phase.

  • Add 300uL of 40C TNES-6U back extraction buffer per 1mL of Trizol to the organic

phase and interphase

  • Shake by hand to mix then incubate at room temperature for 10 min.
  • Spin at 18,000xg for 15min at 4C in a fixed angle table top centrifuge
  • Transfer the aqueous phase to a clean tube (DNA) and discard the interphase (Protein)

and the organic phase

  • Add an equal volume of isopropyl alchohol to the aqueous phase then incubate at -80C for 2hr
  • Spin at 18,000xg for 15min at 4C then discard the supernatant

DNA Wash

  • Wash the pellet 3 times with 70% ethanol (incubate 3min, spin 18,000xg for 5min)
  • Air dry on bench for 5-10min or until liquid is not visible

Redissolve DNA

  • Redissolve as described above, or in 300-500uL of 65C Low EDTA TE buffer pH 8.0 (Affymetrix)

Notes

See also

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