TRIzol® Reagent is a solution of phenol and guanidine isothiocyanate for the extraction of total RNA from cells and tissues. TRIzol® Reagent performs the extraction of RNA from cells by disrupting cells and dissolving cell components while preserving the integrity of the RNA. RNA extraction by TRIzol® represents the first step of RNA isolation. The subsequent addition of chloroform followed by a centrifugation separates an organic phase and an aqueous phase containing the RNA. Then, the addition of isopropyl alcohol to the aqueous phase precipitates the RNA which can be washed with 75% ethanol and finally diluted in water. RNA isolation using TRIzol® is efficient from 5x106
cells, or 50 mg to 1 g of tissue from various origins (human, animal, plants, etc.). The isolated RNA can be used for Northern blot analysis, dot blot hybridization, poly (A)+ selection, in vitro
translation, RNase protection assay, molecular cloning and RT-PCR.
I use TRIzol® to isolate RNA from mammalian cells for analysis via RT-PCR. The cells I use grow in a monolayer. The protocol recommends using 1mL/10cm2, regardless of the number of cells. Calculation of the required mL of TRIzol® according to the surface can give decimals numbers. All the volumes of chemicals added at the following steps are based on the initial volume of TRIzol®. Thus, in order to keep the method easy and efficient, I do not perform a calculation. Instead, I use a round number of mL of TRIzol® that covers the entire surface of the dish.
RNA extraction by TRIzol® is efficient and the RNA is free of protein and DNA contamination. However, it is not rare when 2 strictly identical RNA isolations give different A260/A280 ratios, suggesting that the reliability of the method is not irreproachable.
This method allows quick and simultaneous RNA isolation of a large number of samples. Isolation from 10 to 15 samples takes no longer than 1 hour. Nevertheless, this method counts too many steps, increasing the risk of RNA contamination. TRIzol® is actually used for the RNA extraction, which represents the first step of the RNA isolation. Then, all the others chemicals can be from various origins, diminishing the consistency of this method. A complete kit of RNA isolation using the TRIzol® Reagent could be a solution to improving the reliability and the consistency of this method.
Finally, TRIzol® and some of the other chemical solutions required for the RNA isolation are hazardous and their vapors can be dangerous. Almost all the RNA isolation process must be done in a fume hood, affecting the convenience of this method.