RNA extraction biochemistry (the TRIzol aka TRI aka guanidinium thiocyanate-phenol-chloroform way)

We can get proteins to separate from nucleic acids by denaturing the proteins w/chaotropes like guanidium thiocyanate & phenol. Proteins are only water-soluble because they fold up so that the non-polar, hydrophobic, parts are hidden inside the protein & the polar, hydrophilic parts are sticking out towards the water. Denatured proteins (w/their exposed but coated nonpolar parts) & lipids prefer to dissolve in a solvent that’s more like them, one that’s less polar - they’re more soluble in the organic phase than the aqueous phase, so they move there. Things that are insoluble in the aqueous phase can move to the organic phase or, if they don’t like either or can’t decide, they can just hang out as “gunk” at the “interphase”⠀ 
full text: bit.ly/trizolRNAextraction
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What about the nucleic acids? Depends on the pH. You have a couple of options. If you want both DNA & RNA, use the phenol at a neutral pH. At this pH, both remain in their soluble forms like they are in your cells. This is what’s used in traditional “phenol-chloroform” extraction. But if you only want the RNA, you can use the phenol at a lower (more acidic) pH. DNA’s bases start picking up some of those extra protons floating around → get protonated → t disrupts their base-pairing (binding between strands) → causes them to denature.  
 
A main reason nucleic acids are usually water-soluble is that they have negatively-charged phosphate groups. But, at low pH, these get “cancelled out” by the positive charge of the protonated bases so the molecule becomes less negatively-charged (less anionic) overall, so it prefers the organic solvent. RNA’s bases can do this too, but RNA has those additional hydroxyl (-OH) “legs” that keep them more polar & happier w/water. If you’re using commercial TRIzol, you don’t get to choose → it uses an acidic pH, so the DNA & RNA will split up. If you are using another phenol:chloroform mix, they sometimes come with a buffer you can add to change pH if you want.  
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First you need to homogenize your cells - if you’re working with tissues, you’ll have to grind them up first. But for cells you can just add the TRIzol directly to it. ​It has guanidinium thiocyanate (that’s our chaotrope) and the phenol (our less-polar part) but not the chloroform (our much-less-polar part). It also has ammonium thiocyanate as well as (probably) sodium acetate to keep the pH low. Add this to your cells & mix really well so that all the cells break open & the denaturants have a chance to reach all of the molecules & denature them. ⠀ 
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Then, once they’ve done their job, add chloroform & mix really really well. Phenol & water have really similar densities, which can make them harder to cleanly separate. So you add chloroform which mixes with phenol & is much denser → ensures a sharp separation with the organic phase on the bottom.⠀ 
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But to get that nice sharp separation, you need to centrifuge the mix. Spin it really fast so the heavier organic phase gets pulled to the bottom. If you’re using commercial TRIzol this bottom, organic, phenol-chloroform layer will be pink, but that’s just a dye.⠀ 
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Now very carefully remove the top, colorless, aqueous layer containing the RNA & transfer it to a new tube - it helps to keep the tube at an angle after you pull it out of the rotor. Don’t touch the interface (that gunky separating line) or the organic phase (the pink stuff) - don’t worry - you can always come back & re-extract to get any leftovers but you don’t want to get the DNA or proteins into the aqueous phase. ⠀ 
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Now you need to separate the RNA from all the salts and any lingering phenol. You can do this by selectively precipitating the RNA by adding isopropanol which lowers the dielectric constant of the solvent. This is a fancy way of saving that it reduces shielding around charges so that the positive salt cations can see the negative phosphates and bind to them → neutralizes charge → nucleic acids become less soluble & they don’t have a better solvent alternative to flee to, so they cling to each other → precipitate.⠀ 
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Separate this precipitate by centrifuging again to pull the precipitate to the bottom. Then remove the liquid & toss it (actually you should probably keep it just in case (and you can actually “back-extract,” basically start over with that part in case there’s still RNA in there you can add in) but your RNA should NOT be in there at this point). ⠀ 
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Then resuspend the pellet (which should be small & “gelly” & reprecipitate it . Then, do an ethanol wash to remove extra salts. Resuspend the pellet in 70-80% ethanol - salts are more soluble in ethanol than in isopropanol, which is less polar, so they switch from preferring to bind RNA over solvent to preferring to bind solvent over RNA. But the RNA isn’t content with ethanol, so it remains insoluble. Then centrifuge again & remove the supernatant. ⠀ 
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Then let it air-dry so ethanol evaporates. Now, finally, you can redissolve the RNA pellet.

[@saavanvanced2781]

Best explanation I've ever gotten!!!..very detailed ... thankyou ao much for this effort

[@thebumblingbiochemist]

Glad it was helpful!

[@rogsang9764]

I´m trully grateful for wath you are doing, everytime i dont understand a step in a biochem protocol i came here to solve my doubts, thanks :D

[@thebumblingbiochemist]

So glad you find them helpful!

[@renatagalleta4118]

Gracias por un video tan explicativo y mas que nada por colocar subtitulos!!

[@julianbarron1680]

Thank you a lot! I love your explanations 🖤

[@thebumblingbiochemist]

Thank so much! Happy to help

[@paaamm007]

I love you! You are saving my life.

[@thebumblingbiochemist]

Happy I can help!

[@rafaelcorea3695]

Thanks for the video

[@user-bv7tc1wj1d]

thank u so much !!! Finally this video has subtitle, so i can realize the experiment

[@thebumblingbiochemist]

happy I could help!

[@user-ci7fe1jj8x]

Thanks

[@ricsstudio8116]

thankyou ,very informing

[@yuca_scholar]

this is so good thank you

[@thebumblingbiochemist]

Thanks! Glad it helped!

[@kelvinxu3058]

Great 👍

[@thebumblingbiochemist]

thank you!

[@mosyoutubeanimals]

Great video! I have a question: why do we heat up the elution buffer?

[@thebumblingbiochemist]

Thanks! I think it's just because it will help things dissolve

[@cpard2d2]

Thanks for the in depth explanation, can we do this from blood? I´ve only done blood extractions with columns so I'm not sure.

[@thebumblingbiochemist]

Yes you can, but I've never done that personally. Good luck!

[@cpard2d2]

@@thebumblingbiochemist Thank you! and congrats on your biochemistry classes! you really have the talent to make these things easy and interesting.

[@thebumblingbiochemist]

thanks so much!

[@heatherpeng7437]

Hi, thanks for the helpful video!! What kind of mask should we wear when performing this kind of experiment? thank you!!

[@thebumblingbiochemist]

You should just work in a fume hood if possible and dispose of the trizol waste according to your institution's safety protocols

[@arjunprashanth9756]

Hey,is RNA extraction possible through just the phenol chloroform method?

[@thebumblingbiochemist]

Yes

[@whiteblack1994]

How can I preserve whole blood samples before RNA extraction??

[@thebumblingbiochemist]

I don't know sorry but I'm sure you can probably find some protocols with some Googling. That's what I'd do. Good luck!