TRIzol Reagent From Invitrogen

We are currently living in the post-genomic area. The human genome project indicated that the difference between ape and Adam was not lying simply in the number of genes, but rather in multiple products of these genes (mRNA) and the complex regulation of their expression. The importance of that regulation was most recently recognized by the Nobel Prize Committee, by awarding the ultimate prize in science for the discovery of the mechanism of regulation of one RNA species (mRNA) by another (siRNA). Thus, what could be now more important than isolation of high quality RNA? Fortunately, you can isolate it with ease using the TRIzol reagent.

This mixture of phenol, guanidine isothiocyanate and other compounds was developed by Piotr Chomczynski in the 80-ies, and since then it has been widely used to isolate RNA from tissues and cells of human, animal, and plant origin. The method is simple and quick, gives high yields, allows isolation from various amounts of sample and delivers high quality RNA. Importantly, TRIzol sufficiently isolates not only RNA of high molecular weight, like mRNA, but also low molecular weight RNA including microRNA.

For a decade of my scientific career spent at the bench, I’ve been using TRIzol continuously with no failure. To use TRIzol in the most efficient way there are a few things to remember. First, the stopping point can be made conveniently just after homogenization, which is very useful when working with a large number of samples. You can thus break your isolation into two steps: homogenization and further isolation, storing homogenates between the steps at -80ºC. Second, you do not have to limit yourself to RNA isolation. You can use the same sample to isolate sequentially high quality DNA and protein. DNA can be of interest as it can serve as a normalization tool for the amount of RNA isolated or allow for computation of the number of cells used (with the average of 6 pg DNA in one mammalian cell). Alternatively, you can use yield charts provided by Invitrogen. Protein isolation can be useful if determination of the message and its product in the same sample is important. Third, a little practice is required in removing the RNA-containing upper phase without the interphase. Tilting the tube at a 45º angle can be a useful trick here. Fourth, the RNA pellet can be barely visible, especially when using very small samples. Use of GlycoBlue or a similar pellet visualization aid can be of help. Fifth, don’t let your RNA pellet overdry (even if air drying). Monitor the pellet appearance and immediately when it becomes of clear/opal color and jelly-ish texture and is not surrounded by the remaining ethanol – redissolve it. Ressuspending overdried RNA pellets is a truly Sisyphean task. Six, remember that nucleic acid purification index (A₂₆₀/A₂₈₀ ratio) varies depending upon a diluent (e.g. water: 1.8 = TE: 2.2). Lastly, if you are working with liquids, you should probably consider using TRIzol LS (Liquid Samples) which is simply a little bit more concentrated to allow a smaller amount of reagent to be used with liquid samples.

The only criticism about TRIzol one may have is that working with it needs caution. Exposure of the skin to concentrated phenol solutions causes chemical burns which may be severe. Splashes should be washed off with polyethylene glycol, while larger amounts should be immediately rinsed with copious amounts of water followed by visit to the ER, particularly if your skin was exposed to the phenol/chloroform mixture.

TRIzol is also a little mnemonic (for these of you who love puzzles): TRI stands for Total RNA Isolation but also for ability of simultaneous TRIple isolation of RNA, DNA and protein. In summary, I would highly recommend using TRIzol to isolate RNA for TRIzol’s simplicity of use, high yield and high quality of isolated RNA. Let’s then, while pursuing our quest to discover the gene of soul, isolate in the meanwhile high quality RNA.