Following cell disruption, most methods involve a deproteinisation stage.

This can be achieved by one or more extractions using phenol or phenol/chloroform mixtures. On the formation of an emulsion and subsequent centrifugation to separate the phases, protein molecules partition into the phenol phase and accumulate at the interface. The nucleic acids remain mostly in the upper aqueous phase and may be precipitated from solution using isopropanol or ethanol.

The homogenate is separated into aqueous and organic phases by addition of bromochloropropane (or chloroform) followed by centrifugation (see sidebar, Advantages of Using Bromochloropropane Instead of Chloroform). RNA partitions to the aqueous phase, DNA to the interphase, and protein to the organic phase.

RNA partitions to the aqueous phase, DNA to the interphase, and protein to the organic phase. This highly reliable technique performs well with both small and large quantities of tissues and cultured cells (e.g., samples larger than ~5 mg tissue or 5 x 105 cultured cells) (Figure 1). RNA isolated using TRI Reagent is suitable for use in real-time and end-point RT-PCR, amplification/labeling for microarray analyses, in vitro translation, cDNA synthesis, Northern blotting, and RNase protection assays. RNA preparations isolated using bromochloropropane often do not require an additional DNase treatment, but Ambion's TURBO DNA-free Kit can be used to remove residual genomic DNA before RT-PCR assays. In addition, PCR, Southern blotting, and cloning can be performed using DNA, and denaturing polyacrylamide gel electrophoresis, and Western blot assays can be performed using protein isolated with TRI Reagent.

A complete, ready-to-use reagent for the isolation of total RNA or the simultaneous isolation of RNA, DNA, and protein from a variety of biological samples. • Simple—A single reagent for isolation of DNA-free RNA, RNA-free DNA, and protein • Productive—Obtain higher yields than with traditional guanidine thiocyanate or cesium chloride methods • Versatile—Works with many sample sources, including human, plant, yeast, bacterial, and viral samples • Flexible—Easily scalable depending on starting material and desired yield • Now incorporated into our RiboPure™ and MagMAX™-96 for Microarrays RNA Isolation Kits TRI Reagent® starting material can be either fresh or frozen tissue, or cell samples of almost any size— the protocol is easily scalable. This RNA isolation method is well-recognized and referenced in numerous publications, a testament to its reliability. TRI Reagent® combines phenol and guanidine thiocyanate in a monophase solution to facilitate the immediate inhibition of RNase activity. Biological samples are homogenized or lysed in TRI Reagent®; the subsequent addition of bromochloropropane or chloroform results in the separation of the homogenate into aqueous and organic phases. RNA partitions to the aqueous phase, DNA to the interphase, and proteins to the organic phase. The RNA can then be precipitated from the aqueous phase with the

addition of isopropanol. hybridization assays. The isolated RNA is suitable for any downstream application. amplification for array analysis. PCR. The entire protocol can be completed in approximately 1 hour. Isolated proteins can be used in SDS-PAGE and Western blots. Southern blotting. and cloning can be performed using DNA isolated with TRI Reagent®. or in vitro translation. . including RT-PCR.