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.
Update 2022: Balta and me have published this protocol in Current Protocols. It is open access, and now you can cite the method if you use it! Here is the link.
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
postharvestcentral 
Pingback: RNA isolation with homemade TRIzol reagent | left-leaning scrutinizer
Hi Julian, that’s a lot of Trizol! How long are the aliquots stable once made?
LikeLiked by 1 person
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
LikeLike
Thanks Julian, is DEPC treated and autoclaved glassware good enough to remove residual RNAse contamination, or do you go for anything more rigorous?
LikeLike
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 🙂
LikeLike
Pingback: Dense Phase Separating Gel / Homemade TRIzol combo – Pipette Jockey
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
LikeLike
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!!
LikeLike
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?
LikeLike
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.]
LikeLike
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
LikeLike
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!
LikeLike
Could you tell me the brands and references of the other products you use? Thank you very much in advance.
LikeLike
The chemicals are all standard lab chemicals, for the Phenol I even used a kosher food ingredient version, only 50€ and it worked great! (https://www.sigmaaldrich.com/catalog/product/aldrich/w322318?lang=en®ion=NL).
LikeLike
I want to extract RNA from filamentous fungi, will this method can work for me or i have to add some additional steps
LikeLike
The cell wall sugars may cause trouble, perhaps try it with a few samples, and also the method using SDS in combination with phenol first: https://wp.me/p2wVnD-hF
LikeLike
Please I want to extract RNA from enteric viruses in water. Will this method work?
LikeLike
I am not sure, with low expected yield this may not be the best method.
Can you let me know if you try it out?
LikeLike
Be careful with the use of phenol when it comes to viruses having a protein (Vpg) covalently linked to their genome like Noro- or Enteroviruses. Due to the Vpg the nucleic acid partitions into the organic phase and is depleted from the aqueous phase.
LikeLike
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!
LikeLike
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!
LikeLike
Dear Julian,
Good day and I hope this message finds you well.
When I add sodium acetate to my lysis buffer, the RNA extraction becomes very difficult.
Besides sodium acetate, do you have any other alternative to precipitate RNA in lysis buffer?
Thanks,
Icarus
LikeLike
Dear Icarus,
I am not sure of other compounds would be possible to use, I have not tried. Perhaps you could try KAc, pH 5? NaAc’s role is to buffer the pH to 5.0. Did you use premade 3 M NaAc pH 5 to add to the solution, or did you add the powder directly? That might make a difference. Please let me know if it improves, and how you did it
LikeLike
Dear Julian,
Good day. Do you have any email through which I can reach you?
Thanks,
Icarus
LikeLike
Dear Icarus, you can contact me through the Wageningen university page, I’ll reply by email (sorry I just don’t want to be spammed by bots, so I rather don’t post my address here)
https://www.wur.nl/nl/Personen/Julian-dr.-JC-Julian-Verdonk.htm
LikeLike
Dear Icarus,
Thank you very much for sharing this protocol. We tried it and the extraction is working perfectly. However, we have some trouble with the stability of the solution. We stated a separation of the two phases (aqueous phase and phenol). Is that normal? We tried Triton X 100 and Sarcosyl as emulsifier without success. Thank you very much in advance!
LikeLike
Dear Myriam,
the Trizol should not have a phase separation, perhaps you used buffer saturated phenol to prepare it? It is important to use crystallized phenol (is also much cheaper), you should have no problems then… Hope this helps
LikeLike
Dear Julian,
Good day. I am working on RNA extraction (from vtm vairal) 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 Vtm environment contains small amounts of virus. 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?
Hope to hear from you soon
LikeLike
Dear Julian,
Greetings!!!
I have a question regarding RNA extraction, Such as-
Viral RNA extraction from Serum/Plasma through homemade protocol.
Human blood Total RNA extraction through homemade protocol.
my question is that mostly protocols are published and mentioned as on tissue for total RNA and Viral. Hence, can we apply these protocol for all kinds of RNA. Mostly, Scientists suggest to addng Carrier RNA (Glycogen) during or prior to precipitation.
Kindly reply if you can.
I shall be highly grateful to your help.
Best regards.
LikeLike
Dear Sandeep, I have no experience with these kind of extractions, but I would try the suggestion, that sounds feasible. Hope this helps, good luck
LikeLike
Hi julian,
So quesiton about the phenol. It must be crystalline form? And this is absolutely safe if you melt it in a water bath? I have 250 g crystallized phenol from VWR life science. And if fully melted, you don’t add any water or buffer? I’m also guessing after adding all of the reagents (Amm. thio., Amm. guan, glycerol, phenol, and sod. acet), you just fill the rest with water?
Just want to clarify this before actually making it,
thank you,
Jon
LikeLike
Dear Jon, if you order a glass bottle, and put it closed in a water bath at ~50 C, the crystals will melt and you can pour it into a vessel that contains all the other chemicals. please check our recent paper for details (basically the same info as on this page, but maybe it will help): https://currentprotocols-onlinelibrary-wiley-com.ezproxy.library.wur.nl/doi/full/10.1002/cpz1.351
LikeLike
Hello sir!
Can u plz tell me which homemade things can we use to extract RNA from different tissues without using trizol reagent ?
LikeLike
Dear Hussain, if you cannot make the Trizol, there is another method, using citric acid and sodium citrate, I have a post about that one as well: DNAse-free RNA isolation (for qRT), the Citrate-Citric Acid method
Hope it works!
LikeLike
Hi Dear JC (Julian) Verdonk!
Firstly, Many thanks for sharing the protocols with us. However, I am facing an issue while isolating RNA, I always get the DNA contamination along with isolated RNA even after trying half of the upper aqueous layer.
Can you please guide me any possible solution to avoid this contamination?
Looking forward to hearing back from you.
Best
LikeLiked by 1 person
Dear BK, possibly the pH of the aqueous layer got too high, this can lead to the co-extraction of genomic DNA. What you could try for this is to reduce the amount of tissue used in the extraction. Sometimes the tissue itself can increase the pH of the extraction buffer. Hope ths helps, please let me know if it worked!
Cheers, Julian
LikeLike
Hi, dear JC (Julian) Verdonk!
First of all, thank you very much for sharing the protocol with us. I would like to ask, is this formula suitable for extracting mammalian cells or tissues? (e.g. human, mouse or rat?)
LikeLike
Hi, it should work, plant cells are much more recalcitrant than mammalian cells , so maybe it works better. If you try ig out,can you let us know if it worked?
LikeLike
Thanks, I will try and reply.
LikeLike