Extracting RNA without the help of a kit isn’t as easy, but you can do it! It’s not just toilet paper and Purel – RNA extraction kits are in high demand, as the first step of testing for the novel coronavirus is isolating the viral RNA from a patient sample. At times like this it’s great to know how to do it the “old-school” way. 

With the (normally) ready ability of RNA extraction kits like those made by QIagen, you might not know the attraction of phenol-chlororm RNA EXTRACTION. Actually it’s not that fun – but let’s try to make it fun! Let me try to explain how TRIzol provides a way to extract and purify RNA! TRIzol’s actually a brand name, but it’s harder to find a pun that works with its “generic” name -> acid guanidinium thiocyanate–phenol–chloroform extraction (sometimes aka AGPC). Less fun name but it has published protocols that don’t have any “proprietary ingredients”

EXTRACTION works by separating molecules based on their SOLUBILITY (and insolubility) in different liquids. It’s kinda like having a mixture get a “divorce” and letting the “kids” decide which parent’s house they want to live in after the split – in our case they can choose between an aqueous (water-based) “phase” or an organic, phenol-chloroform “phase” which don’t mix with each other, so will separate into layers. 

Being soluble means that each copy of a molecule has its own full coat of solvent. Most of the time in biochemistry, the solvent we’re talking about is WATER. Our bodies are mostly water, so if we want to study what goes on in our bodies, we use water-based solutions, and we call such water-based solutions AQUEOUS

It’s not just for the sake of “authenticity” that we study compounds in water. Because most molecules in our body have to work in a watery environment, they’ve evolved to function optimally in that environment, which involves being maximally soluble. 

If something’s NOT soluble, it means that the solute molecules (things you’re dissolving) would rather bind to each other than to the solvent molecules (thing in which you’re dissolving) &/or the solvent molecules would rather bond to each other than to the solute.

If molecules bind to each other instead of the solute, they can clump up – aggregate – as precipitate. This maximizes their contact w/each other & minimizes their contact w/the solvent, but it also makes them non-functional. You definitely don’t want this occurring in your cells, so you need the molecules to be soluble. 

But there are solvents other than water, and some molecules like some solvents better than others, largely due to their charge distributions & if you give them options they can choose a different solvent instead of just panicking and clumping up.

There are parts of your body, like your cell membrane that are “fatty,” like the lipid membranes surrounding your cells, so the molecules that work there (at least the membrane-embedded parts) have to be fat-soluble, so they go through special processing steps to ensure they don’t clump. 

So, in our bodies, precipitation is a bad thing, but in the lab we can exploit different molecules’ different abilities to clump under different conditions to remove things from solution that we don’t want in there. Or to remove things from solution that we want so we can then remove all the stuff we don’t want in the liquid.

Some types of precipitation are “reversible” – remember, you’re not breaking any strong covalent bonds, not “cutting any chains” just unspooling the yarn balls. So if you take something that’s precipitated and put it back in the solvent it likes, it might redissolve and refold.

Problem is, folding complex things like proteins is a complex process that often requires the help of other proteins called chaperones (which is why we still can’t predict exactly how a protein will fold based on its sequence alone). So they often can’t re-fold properly.

BUT DNA & RNA have more “simple” structures that can re-fold. So we can take precipitate the nucleic acids, remove the other stuff, and then redissolve the nucleic acids, heat up a bit, cool them off, & they’ll “anneal” back to their original shapes. 

Say you have some cells in a dish and you want to study the RNA they contain. First you have to break the cells open (lyse them) to release the intracellular (cytoplasmic) contents. But a LOT of this stuff you’re left with is PROTEIN. You’re interest in the nucleic acids, so you need to remove the protein, especially the NUCLEASES – enzymes that chew up DNA & RNA.

We can get proteins to separate from nucleic acids by denaturing them with CHAOTROPES like GUANIDINIUM THIOCYANATE & PHENOL. Proteins are only water-soluble because they fold up so that the water-hating (hydrophobic)(nonpolar) parts are hidden inside the protein and the water-loving (hydrophilic)(polar) parts are sticking out towards the water.

CHAOTROPES get them to unfold “loosening up” the water network surrounding the proteins 👉 now binding to water is a less “exclusive club” so the water starts to bind parts of the protein that are more hydrophobic -> causes the protein to unfold but stay soluble – and also solubilizes the lipids in the lipid membrane – splits them apart, breaking open (lysing) the cell. 

And once they’re soluble, they’re free to choose a solvent if there are options. The denatured proteins, with their exposed but coated nonpolar parts and the 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. So, things that are insoluble in the aqueous phase can move to the organic phase (lipids also take this option) or, if they don’t like either or can’t decide, they can just hang out as “gunk” at the “interphase”

What about the nucleic acids – where will they go? Depends on the pH. You have a couple of options, depending if you want the DNA, the RNA, or both. If you want both, use the phenol at a neutral pH. At this pH, both DNA & RNA 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 -> this disrupts their base-pairing (binding between strands) -> causes them to denature. And the negative charge of the phosphates in the backbone gets “cancelled out” by the positive charge of the protonated bases so the molecule becomes less – charged (less anionic) overall, so they prefer the organic solvent. RNA’s bases can do this too, but the RNA has those additional hydroxyl (-OH) “legs” that keep them more polar & happier with 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. 

How do we do it? 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. TRIzol has the guanidinium thiocyanate and the phenol but not the chloroform. It also has ammonium thiocyanate as well as (probably) sodium acetate to keep the pH nice and 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 and denature them. 

Then, once they’ve done their job, add chloroform and mix really really well. Why? Phenol & water have really similar densities, which can make them harder to cleanly separate – especially since, if there’s a lot of stuff in the aqueous phase it could become denser than the phenol and end up on the bottom, not the top where you expect it. So you add chloroform which mixes with phenol and is much denser -> this ensures a sharp separation with the organic phase on the bottom

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.

Now you very carefully remove the top, aqueous layer containing the RNA and transfer it to a new tube. Don’t touch the interface or organic phase – 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. 

Now you need to separate the RNA from all the salts and any lingering phenol, which you can do by selectively precipitating it by adding ISOPROPANOL (aka isopropyl alcohol or propan-2-ol). The isopropanol works by lowering the dielectric constant of the solvent – it reduces shielding around charges so the + salts can see the negative phosphates and bind to them -> neutralize charge -> nucleic acids become less soluble & they don’t have a better solvent alternative to flee to, so they cling to each other -> precipitates

Separate this precipitate by centrifuging again to pull the precipitate to the bottom. Then remove the liquid and 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). 

Then resuspend the pellet (which should be small and “gelly” and reprecipitate it – didn’t I just do that? – yep, hence the “re-“ part – might seem like a pain, but increases the purity! – you want to make sure you remove any phenol.

Then, you do an ethanol wash to remove extra salts. You resuspend the pellet in 70-80% ethanol. you want to coat it in ethanol as much as possible but you’re not re-dissolving it, more like just breaking up the big clump into smaller clumps because what you’re trying to get to dissolve at this point isn’t the RNA, it’s any lingering salts. These salts are more soluble in ethanol than in isopropanol, so they switch from preferring to bind RNA over solvent to preferring to bind solvent over RNA, but the RNA remains insoluble. Then you centrifuge again and remove the supernatant. 

Then you let it air-dry – let the ethanol evaporate. Now, finally, you can redissolve the RNA pellet. You want to make sure you’re dissolving it in RNase-free water so this RNA you’ve worked so hard to purify doesn’t get degraded! 

Now you want to check that it worked – You can use a spectrometer like a “NanoDrop” to check out the absorbance. DNA, RNA, & proteins all absorb UV light – if things absorb visible light, they hide that slice of the rainbow from us so we see the sum of the remaining colors which is no longer white – we see color. But if things absorb light that’s outside of the visible range, we can’t see the change with our naked eyes, but the spectrometer can detect these changes we can’t see -> we can measure absorbance of UV light. Molecules usually absorb a range of wavelengths, giving you an absorption spectrum. DNA, RNA, & protein have somewhat overlapping absorption spectra, but there are characteristic “ratios” that can tell you how pure your samples are. more: http://bit.ly/2CeDj5S

Proteins absorb most strongly at 280nm whereas nucleic acids absorb more strongly at 260. Pure RNA should have an A260/A280 ratio of ~2. Pure DNA should have an A260/A280 ratio of ~1.8. Lower values could indicate you didn’t remove all the phenol and/or protein. This value can also be affected by pH – at a lower pH, the 280 absorbance increases. To get a most accurate measurement, measure at sample aliquot in 1 mM Na2HPO4, pH 7.5

All look good? Go ahead and use it if you’re ready, or, if not store your nice pure RNA at -80°C

If you want the DNA too, you can precipitate it out of that original organic phase using ethanol. This will precipitate the DNA but not the proteins. And if you want to try to rescue that protein, you can precipitate it out of the leftover liquid by adding isopropanol. 

more on chaotropes: http://bit.ly/2R9XkCI

more on dielectric-lowering: http://bit.ly/2DDtyzC

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