TRI Reagent® From Sigma-Aldrich

TRI Reagent® is a quick and convenient reagent for use in the simultaneous isolation of RNA, DNA and protein. Successful isolations from human, animal, plant, yeast, bacterial and viral samples can be obtained. I typically use this reagent for isolation of RNA from tissue culture cells (human and mouse). I only recommend the TRI Reagent® for extraction of total RNA; I have not had good luck with getting good genomic DNA and protein, although Sigma says you should get good yields. I have found that both the DNA and protein are very hard to get back into solution and the DNA needs to be further extracted with phenol:chloroform to remove contaminating proteins.

TRI Reagent® performs the extraction of RNA from cells by disrupting cells and dissolving cell components while preserving the integrity of the RNA. While your sample is in TRI Reagent®, RNases are completely inactivated. RNA can be stored at -80ºC in TRI Reagent® for up to 1 year and RNA will not degrade.

RNA extraction by TRI Reagent® represents the first step of RNA isolation. The subsequent addition of chloroform followed by a centrifugation separates an organic phase (containing protein), an interphase (containing DNA and protein) and an aqueous phase (containing the RNA). After removal of the aqueous phase, the addition of isopropyl alcohol to the aqueous phase precipitates the RNA which can be washed with 70-75% ethanol and finally, diluted in RNase-free water. The pellet is off-white and very visible in 1.5 ml tubes. It is recommended after the 70-70% ethanol wash to allow the pellet to dry. However, do not let the pellet dry too long as the RNA will be hard to dissolve. Also, when the pellet dries, it turns translucent and may become hard to see.

RNA isolation using TRI Reagent® is efficient for up to 1x10⁷ cells or 100 mg of tissue. The isolated RNA can be used for Northern blot analysis, dot blot hybridization, poly (A)+ selection, in vitro translation, RNase protection assay, cloning and RT-PCR. I typically isolate RNA from 3-6 X10⁶ cells (grown in suspension) in 1 ml of the TRI Reagent®. My RNA yields are typically between 40-80 micrograms of total RNA. I have used the RNA for Northern Blots and RT-PCR as well as first strand cDNA synthesis.

The total isolation procedure takes less than an hour to complete and multiple samples can be processed simultaneously. For first time users, I would recommend performing only up to 4 samples, but I have done up to 12 samples at a time.

Finally, TRI Reagent® 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.