TRIzol® Reagent From Invitrogen

TRIzol® is a ready to use reagent, primarily for the isolation of total RNA. The phasic isolation protocol, however, also allows for the isolation of DNA (from the interphase and organic phase) and protein (from the supernatant after precipitation of DNA). The reagent is an improvement of the single-step RNA isolation method developed by Chomczynski and Sacchi. TRIzol is simple to use and suitable for the isolation of RNA from small to large quantities of tissue (50-100 mg to greater than 1 mg) and cells (5 x 10⁶ to greater than 10⁷), of human, animal, plant or bacterial origin. The resultant total RNA is free from both contaminating DNA and protein and suitable for use in downstream applications such as Northern blot analysis, dot blot hybridization, poly(A)+ selection, in vitro translation, RNase protection assays and molecular cloning.

The reagent is a mono-phasic solution of phenol and guanidine isothiocynate. Also required for RNA isolation are chloroform, isopropyl alcohol, 75% ethanol in DEPC-treated water and RNase-free water or 0.5% SDS solution (for re-dissolving the isolated RNA). The protocol supplied with TRIzol is simple to follow and rapid, taking approximately 60 minutes from start to finish. In addition, the simple procedure allows for easy simultaneous processing of multiple samples: I routinely process 20-30 at a given time.

The first step of the protocol requires homogenization of the sample and alternative procedures are given, depending on whether the sample is tissue, cells grown in monolayer or cells grown in suspension. Homogenization allows for the disruption of cells and dissolving of cell components, whilst retaining the integrity of the RNA. There is then an optional isolation step for samples with a high content of proteins, fat, polysaccharides or extracellular material such as muscle, fat tissue and tuberous parts of plants. The homogenized samples are then incubated for 5 minutes at 15-30°C, to permit complete dissociation of nucleoprotein complexes. The next step is phase separation. During this phase, 0.2 ml of chloroform is added per 1 ml of TRIzol, samples are shaken vigorously for 15 seconds and then left to incubate for 2-3 minutes at 15-30°C. Samples are then centrifuged for 15 minutes at 12,000 x g and 30°C. This separates the mixture into a lower red phenol-chloroform phase, an interphase and a colorless upper aqueous phase, which contains the RNA. This aqueous phase must be carefully transferred to a fresh tube, without disruption of the interphase or red lower phase. In my experience, this is the slowest part of the protocol, due to the care required. The volume of the aqueous phase usually comprises 50-60% of the initial volume of TRIzol. If isolation of DNA or protein is required, the lower organic phase must be retained and separate procedures are given. To precipitate the RNA, 0.5 ml of isopropyl alcohol should be added per 1 ml TRIzol, the sample incubated for 10 minutes at 15-30°C and then centrifuged at 12,000 x g at 2-8°C for 10 minutes. The result is a gel-like RNA precipitate pellet. The supernatant is then removed, the pellet washed twice in 75% ethanol by centrifugation at 7,500 x g for 5 minutes at 2-8°C and the final pellet air-dried. RNA can be dissolved in nuclease-free water or 0.5% SDS by passing the solution a few times through a pipette tip and incubating for 10 minutes at 55-60°C.

I use TRIzol routinely for total RNA extraction from cell lines gown in suspension. The total RNA is then used for downstream quantitative RT-PCR of microRNAs. I have found the product ideal for this application. The RNA yielded is always of optimum quality with NanoDrop 260/280 ratios in excess of 1.8 and I routinely obtain a concentration of ~100-400 ng/µl per 10⁷ cells. A number of commercially available RNA extraction kits can be utilized to enrich for small RNAs, including microRNAs. However, there is a risk that these filter based methods, although increasing the yield of microRNAs, may in fact reduce the spectrum. For this reason I find TRIzol perfect, as the phase based extraction eliminates this possibility. In addition, in preliminary experiments, C_T values obtained when amplifying specific microRNAs were optimal for TRIzol based extraction, in comparison to other methods. The reagent is good value for money and quick and easy to use, representing an excellent system for RNA isolation.