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

被引:23
作者
Bachoon, DS [1 ]
Chen, F [1 ]
Hodson, RE [1 ]
机构
[1] Univ Georgia, Sch Marine Programs, Dept Marine Sci, Athens, GA 30602 USA
关键词
prokaryotic mRNA; RT-PCR; preserved bacterial cell; RNA recovery;
D O I
10.1111/j.1574-6968.2001.tb10745.x
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
引用
收藏
页码:127 / 132
页数:6
相关论文
共 26 条
[1]   COMBINATION OF 16S RIBOSOMAL-RNA-TARGETED OLIGONUCLEOTIDE PROBES WITH FLOW-CYTOMETRY FOR ANALYZING MIXED MICROBIAL-POPULATIONS [J].
AMANN, RI ;
BINDER, BJ ;
OLSON, RJ ;
CHISHOLM, SW ;
DEVEREUX, R ;
STAHL, DA .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1990, 56 (06) :1919-1925
[2]   POSTTRANSCRIPTIONAL CONTROL OF GENE-EXPRESSION - BACTERIAL MESSENGER-RNA DEGRADATION [J].
ARRAIANO, CM .
WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY, 1993, 9 (04) :421-432
[3]   MECHANISMS OF MESSENGER-RNA DECAY IN BACTERIA - A PERSPECTIVE [J].
BELASCO, JG ;
HIGGINS, CF .
GENE, 1988, 72 (1-2) :15-23
[4]   In situ reverse transcription, an approach to characterize genetic diversity and activities of prokaryotes [J].
Chen, F ;
Gonzalez, JM ;
Dustman, WA ;
Moran, MA ;
Hodson, RE .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1997, 63 (12) :4907-4913
[5]   Microscopic detection of the toluene dioxygenase gene and its expression inside bacterial cells in seawater using prokaryotic in situ PCR [J].
Chen, F ;
Dustman, WA ;
Hodson, RE .
HYDROBIOLOGIA, 1999, 401 (0) :131-138
[6]   Flow cytometric detection of specific gene expression in prokaryotic cells using in situ RT-PCR [J].
Chen, F ;
Binder, B ;
Hodson, RE .
FEMS MICROBIOLOGY LETTERS, 2000, 184 (02) :291-295
[7]  
CHEN F, 1998, MOL MICROBIAL ECOLOG, P1
[8]   PHYLOGENETIC STAINS - RIBOSOMAL RNA-BASED PROBES FOR THE IDENTIFICATION OF SINGLE CELLS [J].
DELONG, EF ;
WICKHAM, GS ;
PACE, NR .
SCIENCE, 1989, 243 (4896) :1360-1363
[9]   PHYLOGENETIC GROUP-SPECIFIC OLIGODEOXYNUCLEOTIDE PROBES FOR IDENTIFICATION OF SINGLE MICROBIAL-CELLS [J].
GIOVANNONI, SJ ;
DELONG, EF ;
OLSEN, GJ ;
PACE, NR .
JOURNAL OF BACTERIOLOGY, 1988, 170 (02) :720-726
[10]   DETECTION OF MICROORGANISMS IN SOIL AFTER INSITU HYBRIDIZATION WITH RIBOSOMAL-RNA-TARGETED, FLUORESCENTLY LABELED OLIGONUCLEOTIDES [J].
HAHN, D ;
AMANN, RI ;
LUDWIG, W ;
AKKERMANS, ADL ;
SCHLEIFER, KH .
JOURNAL OF GENERAL MICROBIOLOGY, 1992, 138 :879-887