RNA recovery and detection of mRNA by RT-PCR from preserved prokaryotic samples

The effectiveness of maintaining prokaryotic RNA in Synechococcus and Pseud omonas cells, fixed in 96% ethanol, 4% paraformaldehyde, or suspended in RN Alater, and held in cold storage for 3 months was compared. Fluorometric de termination of the RNA extracted from Synechococcus and Pseudomonas cells i ndicated that the cell storage treatments tested were equally effective at maintaining their total RNA content. There was not any detectable decrease in the quantity of RNA isolated from the preserved samples during storage. Intact mRNA transcripts of the RuBisCO (rbcL) and nir genes were detected b y reverse transcriptase-polymerase chain reaction (RT-PCR) from preserved b acterial cells throughout 3 months of storage. In contrast, RT-PCR failed t o amplify the mRNA of the rbcL and nitrite reductase genes in unfixed and/o r unpreserved bacterial samples, suggesting that bacterial mRNA can be well maintained during a prolonged storage when cells are preserved properly. I n addition, RNAlater is a useful reagent for the storage and maintenance of high quality RNA in unfrozen samples. (C) 2001 Federation of European Micr obiological Societies. Published by Elsevier Science B.V. All rights reserv ed.