In austerity times, nothing is in excess. Apart from saving reagents, which can be refilled with extra financial injections, there is a commodity that cannot be easily resupplied – tissue samples!
If, like me, you have experienced the fear of not having enough sample for performing a qPCR, western blot, and conventional PCR from the same sample, you may have resorted to acid phenol-chloroform extraction. With this technique, you can get 3 biomolecules at once by partitioning using their different physical and chemical properties. The substances needed for this isolation are nicely packed together in TRIzol reagent or TRIreagent. However, some say it is cheaper to make homemade extraction reagents.
In the paragraphs you are about to read, I describe the science behind this technique and tips for getting the best out of it!
Preparing the Sample for Acid Phenol Chloroform Extraction
You can use a homogenizer to crash the cells, but I truly recommend pulverizing the tissue with liquid nitrogen and then adding TRIzol. Seems cleaner, kinda. Also, in my experience, you can also add TRIzol to minced tissue and put the tubes on a rotating wheel for half an hour. This will lyse a fair number of cells for isolating enough material.
Separating the Nucleic Acids from One Another with Low pH
After homogenization, add chloroform and centrifuge at full speed. You should see three nice distinct phases – red organic phase, white-ish interphase, and clear aqueous phase.
The science behind it: As Nick explained previously – phenol solubilizes the molecules; proteins (due to many hydrophobic amino acid side chains) are flipped at their hydrophobic side, and remain in the organic phase.
The DNA and RNA have phosphate diesters that are negatively charged at neutral pH. If the pH is 7-8, both nucleic acids will be in the polar, aqueous phase. But we need them separated and we need them intact! This is why the pH is adjusted to acidic (4, 4.5). At this pH, DNA separates to the organic phase and RNA stays largely in the aqueous phase.
The biophysical reasons why RNA preferentially separates into the aqueous phase are complex. RNA has a 2-hydroxyl group, which makes it more polar than DNA. Based on the “like dissolves like” principle, it would preferentially migrate into the aqueous phase.
The 2-hydroxyl group also makes RNA stable in acidic solutions [1] because, under basic conditions, it can become deprotonated to give the 2-alkoxide anion. This anion would be adjacent to the electron-withdrawn phosphorous atom in the phosphodiester moiety and could potentially react with it via nucleophilic attack.
Note that preferential separation of the RNA into the acidic aqueous phase does not occur because of differences between the pKa of the phosphate groups on DNA and RNA or because “RNA is more acidic.” [2]
You can explore some of the other possible reasons for the preferential separation of RNA into the acidic aqueous phase here.
What about chloroform? Phenol alone retains 10-15% of water resulting in an equal loss of RNA; chloroform prevents this since it’s miscible with phenol, but more dense, so it pulls the phenol away from water, making the separation sharper. It also is a good partner with phenol for denaturing proteins and it dissolves lipids.
Critical point: If you use homemade TRIzol, saturate the phenol with water, not buffer. This is because pH is the most important feature of this step. Also, homemade solutions will not yield a red organic phase. To avoid confusion, remember chloroform is much denser than water – the organic phase is always the lower one.
Protocol extension: if you have tissues in an RNA stabilizing buffer, you can add TRIzol and freeze it at -20°C, storing it for several days.
Precipitating RNA from the Aqueous Phase
After you’ve pipetted out the upper aqueous phase, precipitate the RNA by adding isopropanol. Wash the pellet with 75% EtOH and dissolve in DEPC water.
The science behind it: A very important component here (contained in the TRIzol reagent) that enables the isolation of high-quality RNA is guanidine isothiocyanate. It is a chaotropic agent that is one of the most effective protein denaturants. Therefore it efficiently disables RNases. It also helps separate rRNA from ribosomal proteins.
We use isopropanol to precipitate the RNA by a salting out principle. Washing with 75% EtOH removes water-soluble salts while the EtOH part of the wash reagent keeps the RNA precipitated.
Critical point: When you pipet the aqueous phase, be careful not to transfer some organic layer, you’ll contaminate RNA with DNA.
Protocol extension: You can add isopropanol and store the samples at -20°C overnight. This gives you more time to perform DNA isolation, without stressing out over isolating everything at once. Plus, if you leave RNA isolation for the next day (or just for later), you’ll have time to immediately reverse-transcribe it to cDNA, which is much more stable than RNA.
Separating DNA from Proteins without Proteolysis
In the organic phase, you have a mixture of DNA and proteins. Separate the DNA by precipitating it with ethanol. Wash and dry the DNA pellet and resuspend it in water, TRIS, or NaOH solution.
The science behind it: Ethanol only pellets DNA since your proteins are happily dissolved in phenol. Use a sodium citrate/EtOH solution as the first washing reagent. Na-citrate is also a chaotrope; it will remove some of the leftover proteins. Use 75% EtOH for the second wash step which, like in the case of the RNA pellet wash, removes the salts.
Critical point: It is important to let the precipitating agent, EtOH evaporate since the pellet will not dissolve completely if EtOH is not dried out.
Protocol change: you can leave the tubes with the dissolving agent (I’ve used water) overnight at 4°C to ensure better yield.
Precipitating Proteins
Precipitate proteins from the last remaining phenol-ethanol solution with isopropanol, wash the pellet with guanidinium hydrochloride, and dissolve in SDS.
The science behind it: Isopropanol pellets out proteins as it is less polar than ethanol. The guanidinium salts wash denatures proteins as it is a chaotropic agent.
Downside: The only painstaking moment is solubilizing the pellet. This method gives a pretty insoluble pellet. Break the pellet using a water bath or an ultrasonic bath; or both, in my experience. Still, if you heat the pellet with SDS a bit longer and sonicate it, you can get a reasonable amount of protein for WB analysis.
In Conclusion…
The fun fact behind the acid phenol-chloroform extraction method is that it is an extension of a technique described in a 1987 paper that gained over 60000 citations. It is ranked 5th on a list of the most cited articles in the life science field. I sure agree it is a useful technique, do you?
References
- Bernhardt HS, and Tate WP (2012). Primordial soup or vinaigrette: did the RNA world evolve at acidic pH? Biol Direct 7:4
- Thaplyal P, and Bevilacqua PC. (2014) Experimental approaches for measuring pKa’s in RNA and DNA. Methods Enzymol. 549:189–19. doi: 10.1016/B978-0-12-801122-5.00009-X
