Simplify RNA Isolation From Plants And Bacteria

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Simplify RNA Isolation from Plants and Bacteria

• Plant RNA Isolation Aid removes common plant RNA contaminants such as polyphenolics and polysaccharides
• GramCracker™ Reagents lyse Gram positive and Gram negative bacteria quickly and efficiently

Maximize Your Yield of RNA from Plant Tissues

The Plant RNA Isolation Aid is recommended for use with Ambion's RNAqueous™ Kit for purification of total RNA from plant tissues. The reagent contains polyvinylpyrrolidone (PVP), a high molecular weight polymer that binds to and removes contaminants such as polyphenolics and polysaccharides that are commonly present in plant tissues.

Glass fiber filter-based methods of isolation, such as the RNAqueous Kit, are especially suited for plant RNA isolation because they do not include any alcohol precipitation steps, which precipitate many common contaminants found in plant tissues. When used with the RNAqueous Kit, the procedure consists of disrupting the plant tissue in a guanidinium-based lysis solution to which the Plant RNA Isolation Aid has been added. A brief centrifugation then removes the Plant RNA Isolation Aid which has bound any polyphenolic and polysaccharide contaminants. The supernatant is passed through a glass fiber filter under conditions that bind RNA to glass. The filter is washed to remove other contaminants, and the RNA is eluted in a small volume (50 µl or more) of aqueous solution. Figure 1 shows total RNA isolated from several different plant tissues using the Plant RNA Isolation Aid in conjunction with Ambion's RNAqueous Kit.  

Figure 1. Plant RNA Isolated Using the Plant RNA Isolation Aid and the RNAqueous™ Kit. RNA from the indicated plant tissues was analyzed on a denaturing agarose gel.

Bacterial Cell Envelopes Are No Longer Barriers to RNA Isolation

GramCracker Reagents include lytic enzymes and accessory reagents for degrading bacterial cell envelopes to maximize recovery of RNA. The enzymes included are lysozyme and lysostaphin. Lysozyme hydrolyzes the N-acetyl muramic acid linkages in the polysaccharide moiety of the peptidoglycan cell envelope structure in gram-negative and some gram-positive bacteria. Lysostaphin cleaves adjacent glycine residues in the cell envelopes of gram-positive cocci. To use GramCracker Reagents, bacteria are pelleted from liquid culture by centrifugation and treated with one or both enzymes. The cells are then disrupted by vortexing in guanidinium-based lysis solution from your favorite RNA isolation procedure or in a single-phase reagent such as Ambion's RNAwiz™. RNAwiz is especially effective for purifying RNA from microorganisms. Figure 2 shows total RNA recovered from a wide range of bacteria using the GramCracker Reagents in conjunction with RNAwiz. In addition to lysozyme and lysostaphin, the GramCracker Reagents include Lysozyme Dilution Buffer, and an RNase-free solution of EDTA and SDS which may be used in an optional pretreatment step to stabilize intracellular mRNA.  

Figure 2. Total RNA Isolated from Bacteria Using the GramCracker™ Reagents and RNAwiz™. All preps were from 1.5 ml of overnight culture except C. pseudodipthericum, which is from 12 ml of culture. The RNA was analyzed on an EtBr-stained denaturing agarose gel.