RNA isolation with homemade TRIzol reagent

Introduction

My all time favorite RNA isolation, and most used protocol is the TRIzol method. Here I give you the basic protocol, including the recipe to make it yourself, which will save you a lot of money.

Method

  • Grind ~100 mg tissue in LN2 in a 10 ml polypropylene tube and a glass rod
  • Transfer powder to 1.5 mL tube (approx 1/4th of the tube filled with powder)
  • Add 1.2 ml TRIzol (TRIzol is best 1:10 (1 g 10 ml), but 1:5 and 1:2 works also fine)
  • Mix well by inverting, and make sure the mixture is completely molten
  • Shake vigorously 15” Don’t vortex! (shears gDNA, and can cause higher gDNA contamination)
  • Incubate 5’ @ RT
  • Add 0.3 ml CHCl3
  • Shake vigorously 15’’
  • Spin 5’ @ max speed
  • Optional: Perform a Phenol: CHCl3 (1:1) treatment to the (upper) aqueous phase followed by a CHCl3 treatment.
  • Transfer aqueous phase (approx 0.5 ml) to new tube
  • Add 1 volume (0.5 ml) isopropanol to precipitate
  • Mix well by inverting
  • Precipitate 10’ @ RT
  • Spin 5’@ max speed
  • Wash RNA pellet with 70% EtOH
  • Dry pellet
  • Dissolve in appropriate vol. of RNAse free MQ
  • Quantify on gel (1µl) and spectrophotometrically

Preparing TRIzol:

composition:
38%     Phenol (pure phenol from crystals, i.e. SIGMA P1037-500G)
0.8 M  Guanidine Thiocyanate (118.16 g/mol)
0.4 M  Ammonium Thiocyanate (79.12 g/mol)
0.1 M  Sodium Acetate (82.03 g/mol)
5 %      Glycerol

  • It is easiest to buy 500 g Phenol crystals, melt it in a water bath @ ~50 °C and then calculate the rest accordingly with 500 g phenol as 38%.
  • The density of phenol is 1.07 g/cm³, so if 500 g is 38%, this will be 500/1.07 = 467.3 ml, and therefore the total volume will be 467.3 *(100/38)= 1.23 liter.
  • Now you can calculate the rest:
    • 1.23 liter 0.8 M Guanide Thiocyanate: 0.8 M x 118.16 g/mol x 1.23 l = 116.3 g
    • 1.23 liter 0.4 M Ammonium Thiocyanate: 0.4 M x 79.12 g/mol x 1.23 = 38.9 g
    • 1.23 liter 0.1 M Sodium Acetate:1.23 x 0.1)/3 = 41 ml 3 M NaAc pH 5
    • 1.23 liter 5% Glycerol: 1.23/20 = 61.5 ml 100% glycerol
  • Make the aqueous solution (the salts and the glycerol) first, and then when the phenol is molten, add the phenol to it, mix well, aliquote (unless you have 1.23 L bottle), and store it in a cool dark place. Just make sure you dont shoot over te endvolume of 1.23 liter
  • The preparation of 1.23 liter TRIzol will cost you about 10 times less than buying the reagent
  • This TRIzol is not pink, but that is not important, just remember that Chloroform is heavier than water

Remarks:

  • If you expect a lot of contamination of sugars (for example if you are working with fruit) a less-stringent precipitation can help although the yield will also decrease
  • Instead of 1 volume isopropanol precipitate using 0.5 volume isopropanol and 0.5 volume of 0.8 M sodium citrate : 1.2 M NaCl (1:1)
  • There is a better method to get rid of the sugars: the SDS-TRIzol combo method
  • This protocol was previously published on my blog
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20 Responses to RNA isolation with homemade TRIzol reagent

  1. Pingback: RNA isolation with homemade TRIzol reagent | left-leaning scrutinizer

  2. Dr D G David Morgan says:

    Hi Julian, that’s a lot of Trizol! How long are the aliquots stable once made?

    Liked by 1 person

    • julian1973 says:

      Dear David, we make 500 mL aliquots and they are good in the fridge for years (!!). The colour can change a bit, but the RNA extraction still works the same

      Like

      • David Morgan says:

        Thanks Julian, is DEPC treated and autoclaved glassware good enough to remove residual RNAse contamination, or do you go for anything more rigorous?

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      • julian1973 says:

        Phenol keeps RNAse under control, glassware straight out of the cupboard or dishwasher is enough. The water I use for the preparation of Trizol is Milli-Q water without any treatment. RNAse is never a problem with this method up to the isopropanol precipitation. From that point onwards, I use autoclaved eppendorfs and Milli-Q water. Good luck, and please let me know how it went 🙂

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  3. Pingback: Dense Phase Separating Gel / Homemade TRIzol combo – Pipette Jockey

  4. Diogo says:

    Hello Julian,

    That’s great. One question, you mentioned that it won’t be pink. Which colour is it then? Or, do you add any dye to it?

    Best,
    Diogo

    Like

    • julian1973 says:

      Hi Diogo,
      I will be colourless to orange-reddish, depending on the oxidation of the phenol probably. The cheaper the Phenol, the redder, it seems. W322318-1KG-K from SIGMA was very cheap, and worked great. The colour not affect the RNA extraction at all 🙂
      I didnt add any dye, but I am sure someone would know what to add, it should stay in the organic phase, I think.
      Let me know how it works out!!

      Like

  5. Icarus says:

    Is there any chance to replace the phenol with non-alcoholic chemicals? I wonder if we can avoid phenol or other alcohol in RNA extraction?

    Like

    • julian1973 says:

      If you leave a bit of the top layer during the phase separation, there is less change you take phenol to the next step. Yield is quite high, and these days there is no need to have large amount of micrograms anymore. Another good method is CTAB as described in Schaart et al., 2002 [Schaart JG, Salentijn EMJ, Krens FA. 2002. Tissue‐specific expression of the beta‐glucuronidase reporter gene in transgenic strawberry (Fragaria × ananassa) plants. Plant Cell Reports 21: 313–319.]

      Like

      • Icarus says:

        Dear Julian,

        Good day. I am working on RNA extraction (from blood) project in which we use magnetic beads to bind the RNA. The major issues with our current experiment is RNA degradation and unsuccessful removal of all the potential inhibitors from blood proteins. Sadly, our PCR results show that all these inhibitors are not thoroughly removed and it affects the amplification timing too.

        Do you know how we can curb this issues? Well, one of the requirement of my current project is that I am not allowed to use any flammable chemicals like alcohol (ethanol or phenol) or proteinase K.

        Hope to hear from you soon.

        Regards,
        Icarus

        Like

      • julian1973 says:

        HI Icarus,
        I am not sure about work with blood, but if you want to inhibit degrading enzymes using something other than Phenol, simply using a very low pH may also be an option. I am not sure if it is used in the animal field. Here is a description on the use, and it includes references: https://postharvestcentral.com/2016/02/05/dnase-free-rna-isolation-for-qrt-the-citrate-citric-acid-method/
        Good luck!

        Like

  6. Jose M. says:

    Could you tell me the brands and references of the other products you use? Thank you very much in advance.

    Like

  7. Alam Zeb Khan says:

    I want to extract RNA from filamentous fungi, will this method can work for me or i have to add some additional steps

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  8. Fawzeeyah says:

    Please I want to extract RNA from enteric viruses in water. Will this method work?

    Like

  9. Rachel Lippert says:

    Curious if you know the difference between making TRIzol and TRIzol LS? Guessing it’s slightly more concentrated but would love to know the exact recipe!

    Like

    • julian1973 says:

      I dont know what the difference is between the two. I found that for Trizol LS a lower ratio than 1:10 can be used, but that is what I already do with this recipe 1:2 still works ok. If anyone know the difference, please let us know!

      Like

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