TRI Reagent® From Ambion

The TRI Reagent® from Ambion is a ready-to-use reagent for the isolation of total RNA. It can also be used to simultaneously isolate RNA, DNA and protein from all kinds of biological material (such as human, animal, plant, yeast, bacterial, and viral samples). The reagent can be scaled to accommodate various sample sizes and works well with samples larger than 5 mg tissue or 5 x 10⁵ cultured cells. TRI Reagent® contains phenol and guanidine thiocyanate to rapidly inhibit RNase activity. Biological samples are homogenized (we use a Dounce Homogenizer) or lysed in TRI Reagent®. The homogenate is then separated into an aqueous and organic phase by adding bromochloropropane and subsequent centrifugation. RNA will be present in the aqueous phase, DNA in the inter-phase, and proteins in the organic phase. Next, the RNA is precipitated from the aqueous phase with isopropanol (in addition to the manufacturer’s protocol, we also use GlycoBlue (Ambion, cat. no. AM9515) as a carrier. The precipitate is washed with ethanol and finally dissolved in RNase-free water.

In most reverse transcriptase-PCR (RT-PCR) experiments, the RNA isolation is the most critical step. The samples are often small, so only very little RNA is present. Moreover, RNA-degradation is a major concern and all precautions should be taken to prevent this. To obtain sufficient amounts of RNA, an RNase-inhibitor should be present immediately upon grinding/homogenizing the sample, and contamination with RNases in all subsequent steps of the protocol has to be avoided.

We are using this reagent to isolate high quality RNA from the brain of Cotesia glomerata and C. rubecula, minute wasps that parasitize caterpillars. The RNA is used in a RT-PCR to study the learning ability of these insects. We are using 20 brains (approximately 1 mg of tissue) in each experiment in order to obtain a sufficient amount of RNA. After dissecting, the brains from the wasps are immediately placed in a Dounce Homogenizer (glass tube with pestle) which contains 1 ml of the TRI Reagent®. We add GlycoBlue at the precipitation step because the RNA pellet we get is otherwise not visible. The isolated RNA is of high quality and there is sufficient RNA present (approximately 0,5 – 1 µg/20 brains) to serve as a template in RT-PCR. In our case, there is no DNA contamination so there is no need for a DNase step. We have tried other protocols, such as a conventional phenol-based protocol, to isolate RNA but have never found such satisfying results as with the TRI Reagent®.

It’s a fast protocol (1hr.), easy to use yet flexible enough to allow adjustments to match the requirements of each new isolation. To optimize the procedure, it might be a good idea to test your own variables (such as amount of starting material) which each new experiment. Once these are set, you have a fast and reliable RNA isolation method.