RNA isolation with homemade TRIzol reagent

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Introduction

It is weird, but I really love RNA isolation, and have used a lot of different methods over the years, but my all time favorite, and most used is TRIzol. 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.
• The preparation of 1.23 liter TRIzol will cost you about 20 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