Isolation of RNA from tissue and cells is an essential tool to study gene expression. Chomczynski and N. Sacchi developed a single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
1. Based on this method, Invitrogen invented the improved ready-to-use monophasic solution of phenol and guanidine isothiocyanate, Trizol®, for easy and efficient RNA isolation.
Trizol® is available as 100 and 200 ml solution in brown glass bottles. When stored at room temperature, it is stable for 12 months, but Invitrogen recommends storage at 2-8°C. High quality RNA (A260/280 nm, 1.6-1.8) can be isolated from a small (50-100 mg, 5x106) or large amount of tissue or cells (≥1g , >107) from human, animal, plant, yeast and bacteria. Several samples can be simultaneously processed. The procedure is fast, taking about one hour to complete. The isolated RNA, containing a variety of RNA species of different sizes, can be used in downstream application like poly A+ isolation, Northern blotting, in vitro translation, RNA protection assays (RPA) and molecular cloning. For use in PCR, Invitrogen recommends treatment of the RNA with DNase I, if primers are in the same exon. Invitrogen's website provides a table of expected RNA yield per mg tissue. In addition, DNA and protein can be isolated from the same sample, allowing determination of expression levels and normalization of RNA.
Additional Trizol® reagents were developed by Invitrogen for special RNA isolation needs: Trizol® LS for RNA isolation from liquid solutions, Trizol® Max™ Bacterial Isolation Kit (Trizol® and Bacterial enhancement reagent) and Trizol® Plus RNA purification system, which combines Trizol® with spin columns to isolate high quality RNA from especially difficult tissue e.g. fibrous or plants.
As with any RNA extraction, care must be taken to avoid contamination with RNases: use autoclaved plastic tubes or baked glassware, filter pipette tips, autoclaved water treated with DEPC and wear gloves. Pipetting Trizol® should always take place under a fume hood to avoid breathing vapors; wearing gloves and goggles is also recommended to avoid skin burns from phenol spills.
We have routinely used Trizol® to isolate RNA from brains and spinal cords of diseased mice and healthy controls. Samples were snap frozen in pre-weighed tubes and stored at -80°C. Since I wanted to compare several samples, I performed the isolation mostly with 6-8 samples in parallel. After weighing the tubes to determine the tissue weight, Trizol® was added in the recommended volume, usually 1 ml per 50-100 mg tissue and the brains and spinal cords were quickly homogenized with a small probe of a handheld polytron. In order to make sure that the tissue was completely homogenized and to remove excessive fat (fat visible as a top layer), we centrifuged (12,000 x g 10 min) the homogenate and transferred the clear solution to a new tube. After chloroform addition and phase separation, care has to be taken when removing the RNA separated into the aqueous phase (top layer) in order to avoid carrying proteins over from the interphase. I usually left a small volume on top of the interphase and transferred the majority of the aqueous phase to a new tube; later, I could easily remove and discard then the residual aqueous phase with a smaller pipette before isolating the DNA. After adding isopropanol to the aqueous phase (0.5 ml/1ml Trizol®) and also during the ethanol wash, it is important to remember on which side of the tube the pellet is expected to precipitate. The pellet is invisible but can be located either to the side or the bottom of the tube (depending upon the tube position during centrifugation) and can be easily lost while resuspending. It is critical to not overdry the RNA pellet, since it will become difficult to dissolve in water even after an incubation time of 10 min at 55°C. Longer incubation times (>10 min) at 55° C or vigorous pipetting for a longer time to dissolve the overdried pellet might result in a degradation of the RNA. The concentration and state of solubility (RNA with a O.D. <1.6 is only partially dissolved) can be obtained by measuring the RNA at A260/280 nm. In addition, I tested my RNA for degradation by running a formamide gel before using it in the analysis of cytokine levels via RPAs. Sometimes I also isolated DNA and protein, but found it difficult to completely resuspend the protein pellet even after incubation in 1% SDS at 50°C. It was especially difficult with brain and spinal cord samples, since the amount of protein is low, making it not only impossible to analyze the protein concentration in a more dilute sample (to lower the SDS concentration), but also to detect low abundant proteins in the sample in Western blots with immunodetection.
To quickly and reliably isolate high quality RNA and DNA from several samples simultaneously, Trizol® is an excellent reagent. The protocol is easy to follow and allows interruption after the homogenization when samples can be frozen at -80°C, if necessary. Invitrogen's website contains the well-explained protocol with hints for working with Trizol® and RNA and FAQs about the method.
1Chomczynski, P and Sacchi, N, “Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.” Anal Biochem, 162(1):156-9, Apr 1987.