RNA extraction using trizol/tri

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== Reagents ==
== Reagents ==
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* TRIzol or TRI reagent
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* TRIzol or TRI reagent  
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: If you want to make your own reagents, see here [[RNA extraction using self-made guanidinium-acid-phenol reagents]]
* 0.8 M sodium citrate / 1.2 M NaCl
* 0.8 M sodium citrate / 1.2 M NaCl
* isopropanol (2-propanol)
* isopropanol (2-propanol)
* chloroform
* chloroform
* 75% EtOH in DEPC H2O
* 75% EtOH in DEPC H2O
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* RNase free water (filtered or DEPC)
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* RNase free water (filtered or [[DEPC]])
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<small>
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<pre>
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draw water into RNase-free glass bottles
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add diethylpyrocarbonate (DEPC) to 0.01% (v/v)
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let stand overnight and autoclave
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</pre>
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</small>
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=== Mixing your own "TRI" reagent ===
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So called Solution D (based on Chomczynski and Sacchi [[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=2440339 1987]/[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=17406285 2006]) is:
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* 4 M guanidinium thiocyanate
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* 25 mM sodium citrate
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* pH 7.0
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* 0.5% (wt/vol) N-laurosylsarcosine (Sarkosyl)
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* 0.1 M 2-mercaptoethanol
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Prepare stock with:
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* dissolving 250 g guanidinium thiocyanate in 293 ml water at 65 °C
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* add 17.6 ml of 0.75 M sodium citrate, pH 7.0
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* 26.4 ml of 10% (wt/vol) N-laurosylsarcosine
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(stored <3 months at room temperature)
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Working solution from stock:
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* add 0.36 ml of 98% 2-mercaptoethanol to 50 ml of stock solution
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(store <1 month at RT)
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=== Using Solution D ===
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Assuming you start with 10 million cells or 100 mg of tissue (keep cool when possible):
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* add 1 ml solution D (don't linger on this step)
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* transfer to tubes
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* add 0.1 ml of 2 M sodium acetate, pH 4.0, and invert tube to mix
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* add 1 ml water-saturated phenol (never buffered phenol) and invert tube
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* add 0.2 ml of chloroform/isoamyl alcohol (49:1) and shake vigorously for 10 sec
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* centrifuge 20min 10000G 4ºC
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* transfer top aqueous phase into new tube
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* precipitate
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== Steps ==
== Steps ==
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== External links ==
== External links ==
Reagents
Reagents
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* [http://www.invitrogen.com/content.cfm?pageid=469 TRIZOL reagent] (Invitrogen), [http://en.wikipedia.org/wiki/TRIzol TRIZOL on wikipedia]
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* [http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&productID=15596026 TRIZOL reagent] (Invitrogen), [http://en.wikipedia.org/wiki/TRIzol TRIZOL on wikipedia]
* [http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Molecular_Biology/DNA_and_RNA_Purification/Tri_Reagent_RNA_Isolation_Reagent.html TRI reagent] (Sigmal Aldrich)
* [http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Molecular_Biology/DNA_and_RNA_Purification/Tri_Reagent_RNA_Isolation_Reagent.html TRI reagent] (Sigmal Aldrich)
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* [http://www.animal.ufl.edu/hansen/protocols/RNA_extraction.htm RNA extraction with TRIZOL] (Uni Florida)
* [http://www.animal.ufl.edu/hansen/protocols/RNA_extraction.htm RNA extraction with TRIZOL] (Uni Florida)
* [http://www.utm.utoronto.ca/~w3flyma/protocols/Trizol%20Troubleshooting.pdf Troubleshooting guide for TRIZOL extraction] (Uni Toronto)
* [http://www.utm.utoronto.ca/~w3flyma/protocols/Trizol%20Troubleshooting.pdf Troubleshooting guide for TRIZOL extraction] (Uni Toronto)
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* [http://en.wikipedia.org/wiki/Phenol-chloroform_extraction Phenol-chloroform extraction of nucleic acids at Wikipedia]
Tips
Tips
* [http://www.ambion.com/techlib/tn/94/949.html Ambion tips on RNA extraction]
* [http://www.ambion.com/techlib/tn/94/949.html Ambion tips on RNA extraction]
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[[Category:Protocol]]
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[[Category:RNA]]
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[[Category:In vitro]]

Current revision

RNA extraction with TRIzol (Invitrogen product name) or the equivalent TRI (Sigma-Aldrich product name) is a common method of total RNA extraction from cells based on the research of Chomczynski P, Sacchi N. 1987 [1] and reviewed by the authors again in 2006 [2]. It takes slightly longer than column-based methods like RNAeasy but it has higher capacity and can yield more RNA. Along with chaotropic lysis buffers it is generally considered the method that gives the best quality RNA.

Contents

Principle

  • guanidinium isothiocyanate (powerful protein denaturant) -> inactivation of RNases
  • acidic phenol/chloroform -> partitioning of RNA into aqueous supernatant for separation

Note: low pH is crucial since at neutral pH DNA not RNA partitions into the aqueous phase. Check the pH of old TRIZOL/TRI reagents!

Reagents

  • TRIzol or TRI reagent
If you want to make your own reagents, see here RNA extraction using self-made guanidinium-acid-phenol reagents
  • 0.8 M sodium citrate / 1.2 M NaCl
  • isopropanol (2-propanol)
  • chloroform
  • 75% EtOH in DEPC H2O
  • RNase free water (filtered or DEPC)

Steps

cell lysis

Image:Time required.png Cell lysis only takes a few minutes per well, but tissue homogenisation can take 10-20 minutes per sample depending on how tough the tissue is.

  • (PBS wash)
  • add trizol (cell lysis)
1ml / 3.5 cm diameter well (6-well)
5ml / 75 ml bottle
  • homogenise by pipetting several times (mechanic lysis)
alternative for tubes: vortex 1 min
alternative for tissue: grind 1 g tissue in liquid nitrogen in a motar and pestle, put tissue into plastic screw-cap centrifuge tube + 15 ml TRIzol reagent, incubate samples for 5 min at room temp or 60° C (scaled up as needed)
  • (5min at RT for complete dissociation of nucleoprotein complexes)

Image:Pause point.png RNA is stable in trizol which deactivates RNases. You can take a break at this point keeping the sample in trizol for a short time or freezing it for a longer one.

phase separation

Image:Time required.png 15-45 min depending on number of samples and whether an additional chloroform wash is necessary

  • add chloroform (1/5 volume of trizol; e.g. 0.2ml to 1ml)
  • shake for 15 sec (Eccles protocol: do not vortex)
  • incubate 2-5 min at RT
  • spin max. 12000g, 5-15 min, 2-8°C
if centrifugation hasn't been sufficient the DNA-containing interphase will be cloud-like and poorly compacted

Image:Optional step.png If supernatant appears turbid an additional chloroform cleaning step can be inserted here.

  • transfer aqueous upper phase into new tube

Image:Difficult_step.png Take care not to aspirate the DNA-containing white interface. This quickly happens and will lead to DNA contamination in your RNA prep.

TRIZOL phases after chloroform addition
 
TOP    - colourless aqueous phase              (RNA) - 60% TRIZOL volume
MIDDLE - interphase                            (DNA)
BOTTOM - red (organic) phenol-chloroform phase (proteins & lipids)

RNA precipitation and wash

Image:Time required.png 20-40 min depending on number of samples

  • add isopropanol (70% of aqueous phase or 1/2 trizol volume)
  • 0.8 M sodium citrate or 1.2 M NaCl can be added
  • (incubate 10min at RT)
  • spin max g, 10-15 min, 4ºC
  • remove supernatant
  • (alternative RNA precipitation - RNeasy from Qiagen) better than alcohol precipitation for smaller amounts of RNA (less risk of losing a miniscule nucleic acid pellet); also reduces risk of organic solvent contamination

similar kits to RNeasy: MinElute kit, or Affymetrix sample clean-up

RNA wash

Image:Time required.png 15-30 min depending on number of samples

  • wash pellet 70% EtOH (add & vortex briefly)
70% ethanol prepared with RNase-free water

Image:Optional step.png some prefer to wash the pellot more than once with 70% ethanol

  • spin max g, 2-10 min, 4ºC
  • air-dry pellet for 5-10 min Image:Critical_step.png Do not overdry the pellet or you won't be able to redissolve it.

Image:Optional step.png optional add RNase inhibitor

Image:Optional step.png incubate at 55-60 C° for 10 min if hard to redissolve

  • transfer to eppendorf tube
  • spin 4° C, 5 min (to pellet undissolved material)

redissolving of RNA

  • dissolve pellet in 50-100 µl filtered or DEPC H2O (note: DEPC inhibits RT reaction)
  • alternatively, 0.5% SDS

pipetting up and down, heat to 55-60°C for 10 min

Common mistakes

  • use too little trizol; very small volumes are hard to separate and will most likely lead to contamination
  • aspirate some white interphase (DNA) when removing aqueous supernatant (RNA)
  • use phenol/chloroform of the wrong pH (has to be acidic)
  • not working under the hood (phenol is toxic [3], chloroform is a narcotic [4])

See also

External links

Reagents

Protocols

Tips

Personal tools