A quick RNA mini-prep for Neurospora mycelial cultures

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A quick RNA mini-prep for Neurospora mycelial cultures
Lindgren, K.M., A. Lichens-Park, J.L. Loros and J.C. Dunlap Dept. of Biochemistry, Dartmouth Medical School, Hanover, NH 03756.
Most RNA isolation techniques currently in use have been developed for the processing of large quantities of material. These typically involve multiple phenol extractions (Reinert et al. 1981 Mol. Cell Biol. 1:829-836) or guanadinium isothio-cyanate/cesium chloride gradients (Chirgwin et al. 1979 Biochem 18:5294-5299) and can be both expensive and time consuming. Often, however, needs arise where quantitatively smaller amounts of RNA are needed from many different samples, for example, during time series analyses or when screening transfor-mants for expression of a transformed gene. Under such circumstances, existing tech-niques are overly time consuming and yield more RNA than is necessary. The availability of a rapid RNA mini-prep is thus desirable. Such a system has been developed for isola-ting plant RNA (Nagy et al. 1988 Plant Molecular Biology Manual, B4; ed. Gelvin and Schilperoort, Klewer Academic Publishing, pp. 1-29), and we have adapted this procedure for use with Neurospora and, potentially, other filamentous fungi. Below, we describe the use of this procedure with 50 ml mycelial cultures, although we have used in with equal success with 5 ml cultures without scaling down the amounts of any reagents.

The method involves the use of a triphenylmethane dye, aurintricarboxylic (ATA), to protect the RNA. ATA binds irreversibly to RNA and is a potent inhibitor of most nucleic acid binding enzymes (Hallick et al. 1977 Nucl. Acids Res. 4:3055-3064). Thus, RNA made with procedure cannot be used for in vitro transcription or translation or reverse transcription but works fine for RNA/DNA or RNA/RNA hybridizations.

To minimize RNase contamination, all glassware is baked at 182°C for at minimum of four hours. Work with gloved hands. The procedure is as follows:

1. Conidia from slants (grown in 16 x 150 mm test tubes containing 8 ml of solid medium) are resuspended in 50 ml of Horowitz complete medium (Horowitz 1947 J. Biol. Chem. 171:255-262) and the cultures grown overnight with shaking at 30°C. A 50 ml culture typically yields enough RNA for 200 gel lanes (see below), and, as noted, smaller culture volumes may be used.

2. Flat mycelial pads are easier to grind than mycelial balls. Therefore, filter cultures using a Buchner funnel onto Whatman #44 filter paper. Wrap flat mycelial pads in aluminum foil and freeze in dry ice. Do not freeze in EtOH/dry ice bath because alcohol might seep through foil. Pads can be stored at -70°C for at least three weeks.

3. Wash a mortar and pestle thoroughly with warm water and Alconox (Fisher Scientific); cool by filling with liquid N2. Remove frozen, flat mycelia from foil and add it to the liquid N2 in mortar. Add ~0.5 g of baked sand and grind mycelial pad to a fine powder. Add more N2 as needed. Mortar and pestle should be washed after every sample.

4. Working quickly before powder can thaw, pour or spoon ground mycelia into 15 ml round bottom Sarstedt tube (Sarstedt tubes #60.540, Princeton, NJ) containing 8 ml of E buffer at room temperature. [E buffer: 50 mM Tris-Cl pH 8.0, 300 mM NaCl, 5 mM EDTA, pH 8.0, 2% SDS; autoclave and add 1 mM ATA and 14 mM