Simultaneous extraction from bacterioplankton of total RNA and DNA suitable for quantitative structure and function analyses.
The aim of this study was to develop a protocol for the simultaneous extraction from bacterioplankton of RNA and DNA suitable for quantitative molecular analysis. By using a combined mechanical and chemical extraction method, the highest RNA and DNA yield was obtained with sodium lauryl sarcosinate-phenol or DivoLab-phenol as the extraction mix. The efficiency of extraction of nucleic acids was comparatively high and varied only moderately in gram-negative bacterial isolates and bacterioplankton (RNA, 52 to 66%; DNA, 43 to 61%); significant amounts of nucleic acids were also obtained for a gram-positive bacterial isolate (RNA, 20 to 30%; DNA, 20 to 25%). Reverse transcription-PCR and PCR amplification products of fragments of 16S rRNA and its genes were obtained from all isolates and communities, indicating that the extracted nucleic acids were intact and pure enough for community structure analyses. By using single-strand conformation polymorphism of fragments of 16S rRNA and its gene, community fingerprints were obtained from pond bacterioplankton. mRNA transcripts encoding fragments of the enzyme nitrite reductase gene (nir gene) could be detected in a pond water sample, indicating that the extraction method is also suitable for studying gene expression. The extraction method presented yields nucleic acids that can be used to perform structural and functional studies of bacterioplankton communities from a single sample.
Weinbauer
Markus G
MG
Department of Environmental Microbiology, AG Microbial Ecology, GBF-German Research Center for Biotechnology, D-38100 Braunschweig, Germany. wein@nioz.nl
Fritz
Ingo
I
Wenderoth
Dirk F
DF
Höfle
Manfred G
MG
D000818:Animals; D001419:Bacteria / Q000235:genetics*; D016172:DNA Fingerprinting; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000032:analysis; D017753:Ecosystem; D005618:Fresh Water / Q000382:microbiology; D020459:Genes, rRNA; D010933:Plankton; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D012329:RNA, Bacterial / Q000032:analysis / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S