RNA recovery and detection of mRNA by RT-PCR from preserved prokaryotic samples.
The effectiveness of maintaining prokaryotic RNA in Synechococcus and Pseudomonas cells, fixed in 96% ethanol, 4% paraformaldehyde, or suspended in RNAlater, and held in cold storage for 3 months was compared. Fluorometric determination of the RNA extracted from Synechococcus and Pseudomonas cells indicated 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 by reverse transcriptase-polymerase chain reaction (RT-PCR) from preserved bacterial cells throughout 3 months of storage. In contrast, RT-PCR failed to amplify the mRNA of the rbcL and nitrite reductase genes in unfixed and/or unpreserved bacterial samples, suggesting that bacterial mRNA can be well maintained during a prolonged storage when cells are preserved properly. In addition, RNAlater is a useful reagent for the storage and maintenance of high quality RNA in unfrozen samples.
Bachoon
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DS
Department of Marine Sciences, School of Marine Programs, University of Georgia, Athens, GA 30602, USA. dbachoon@arches.uga.edu
Chen
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F
Hodson
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D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D011309:Preservation, Biological; D012329:RNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger