Reagents which are being sold because of their simplicity, ease and convenience constitute a rapidly growing area within the sciences. Ambion’s TRI Reagent® is one such reagent. It can be used for the isolation of RNA, DNA, and/or protein. I have used this reagent to extract, separate and purify RNA, DNA and protein from cell lines (H9, HUT 78) as well as from E. coli and yeast. I have also observed the process from the isolation of mouse tissues.
The TRI Reagent® solution combines phenol and guanidine thiocyanate in a monophasic solution to rapidly inhibit RNase activity. A biological sample is homogenized or lysed in TRI Reagent® solution and the homogenate is then separated into aqueous and organic phases by adding bromochloropropane (BCP) and centrifuging. RNA partitions to the aqueous phase, DNA to the interphase, and proteins to the organic phase. RNA can be precipitated from the aqueous phase with isopropanol, and then washed with ethanol and solubilized. Following removal of the RNA-containing aqueous phase, ethanol is added to the tube and the DNA is pelleted by centrifugation. The protein can then be precipitated from the phenol-ethanol supernatant. After a series of washes, the protein-containing pellet is solubilized in a suitable detergent-containing solvent. It is important to follow the protocol with respect to the number of cells you are using.
The process for the separation, isolation and purification of these macromolecules (DNA, RNA and proteins) is based upon the relative differences in the ratio of their densities, molecular weights and solubilities. Many purification schemes are based upon these characteristics. When I first used the TRI Reagent®, I was skeptical that the mixture would be good with respect to the separation of the macromolecules. I found that the reagent did an excellent job in the separation of proteins from nucleic acid. Previously, I had manually separated each macromolecule independently (i.e one separation for DNA, another for RNA and another for protein). The TRI Reagent® mixes these together to shorten the number of steps.
However, TRI Reagent® does not work well with all cell types. My experience with it is that it works well with cell lines, bacteria and yeast. However, with materials which have significant amount of cellulose and/or plant walls, you many need to change the volumes and/or ratios of EtOH, phenol, ispropanol, and guanidine hydrochloride as well as alter the types of detergents used in order to achieve a good isolation.
I recommend this reagent for its relative ability to lessen and shorten a number of steps in the isolation of DNA, RNA and protein. Use of the TRI Reagent® is relatively easy and reliable. The only caveat is that it not a reagent for all types of tissues and cells.