Plant Genome III Conference Plant Genome III Conference
Construction of cDNA libraries and the development of RNA rotation procedures for plants can be time consuming, require expensive enzymes or mechanical devices to break open cell walls, and often is difficult and frustrating. In this report we describe a simple and reliable system for isolating undegraded plant RNA from any source of tissue. The basic method was recently described to isolate RNA from Staphylococcus aureus and Mycobacterium tuberculosis (A. Cheung, Anal. Biochem. 222:511- 514, 1994). The method uses the new FastPrep(tm) device an extremely rapid reciprocating-motion machine to homogenize plant tissues and lyse cells in a tube containing corarnic/silica matrix or glass spheres and proprietary reagent mixtures to Inhibit nucleases. Plant tissues are placed in a tube with matrix and reagents and processed for an average of 30 seconds, depending on callus type, causing immediate lysis and Rnase inactivation. Lysate is removed from the tubes and the RNA is precipitated with alcohol, washed and dissolved in water or low salt buffer. The procedures require only 15 minutes and up to 12 samples can be processed simultaneously. DNA contamination of RNA samples, as measured by diphenylamine color test is undetectable in most cases. Typical OD260/OD280 ratios are 1.9-2.0 for RNA. We have studied more then 300 different plane and tissues Including flowers, leaves, stems, seeds, fruits, seedlings, and roots. Agarose gels, OD readings, and DNA measurements demonstrate the integrity and purity of RNA preparations. The FastPrep(tm) system consists of the FastPrep(tm) instrument and FastRNA(tm) kits is produced as a joint venture of Savant Instruments, Inc. (Farmindale, NY) and BI0 101, Inc. (Vista, CA). The FastPREP(tm) system is available through BIO 101 and provides a rapid, simple, and cost effective procedure for isolating undegraded plant RNA for cDNA library construction.