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Results for “total RNA”

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At least 145 records · Page 8Linked to original sources
PubMed2000

Whole-cell versus total RNA extraction for analysis of microbial community structure with 16S rRNA-targeted oligonucleotide probes in salt marsh sediments.

rRNA-targeted oligonucleotide probes have become powerful tools for describing microbial communities, but their use in sediments remains difficult. Here we describe a simple technique involving homogenization, detergents, and dispersants that allows the quantitative extraction of cells from formalin-preserved salt marsh sediments. Resulting cell extracts are amenable to membrane blotting and hybridization protocols. Using this procedure, the efficiency of cell extraction was high (95.7% +/- 3.7% [mean +/- standard deviation]) relative to direct DAPI (4',6'-diamidino-2-phenylindole) epifluorescence cell counts for a variety of salt marsh sediments. To test the hypothesis that cells were extracted without phylogenetic bias, the relative abundance (depth distribution) of five major divisions of the gram-negative mesophilic sulfate-reducing delta proteobacteria were determined in sediments maintained in a tidal mesocosm system. A suite of six 16S rRNA-targeted oligonucleotide probes were utilized. The apparent structure of sulfate-reducing bacteria communities determined from whole-cell and RNA extracts were consistent with each other (r(2) = 0.60), indicating that the whole-cell extraction and RNA extraction hybridization approaches for describing sediment microbial communities are equally robust. However, the variability associated with both methods was high and appeared to be a result of the natural heterogeneity of sediment microbial communities and methodological artifacts. The relative distribution of sulfate-reducing bacteria was similar to that observed in natural marsh systems, providing preliminary evidence that the mesocosm systems accurately simulate native marsh systems.

Ecosystem↗
PubMed2002

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.

Animals↗
PubMed1983

Structural proteins and cell-free translation products of total RNA and hybrid-selected RNA from two DNA variants of vaccinia virus.

Two major variants of vaccinia virus, large (L) and small (S), differ by a deletion of 9.7 kilobase pairs. The structural proteins and the translation products of RNA transcribed in vitro from each of these variants were analyzed by gel electrophoresis. Recombinant plasmids were used to select RNA transcribed from the L variant sequences corresponding to the deletion. This RNA yielded translation products indicating that a minimum of 11 polypeptides, including two structural proteins, map within the deletion.

DNA, Recombinant↗
PubMed2000

Isolation and purification of functional total RNA from woody branches and needles of Sitka and white spruce.

The isolation of intact, functional RNA from conifer spp. is not easy, especially from those tissues that are heavily lignified and characterized by a low number of living cells. An efficient procedure for isolating RNA from combined wood and bark tissues of conifers was developed based on a protocol optimized for the extraction of RNA from pollen and one for the isolation of RNA from woody stems. This protocol does not involve the use of phenol, and no ultracentrifugation was required. In addition, the protocol overcame the problems of RNA degradation and low yield due to oxidation by polyphenolics and co-precipitation with polysaccharides, both of which are abundant components in conifer bark tissues. The isolated RNA was of high quality and undegraded as gauged by spectrophotometric readings and electrophoresis in denaturing agarose gels. Quality was further assessed through the subsequent use of the RNA in reverse transcription and RT-PCR, indicating that it could be used for a number of downstream purposes including Northern blot hybridization and cDNA library construction. Using this modified protocol, 80-150 micrograms of RNA was routinely obtained from 1 g of fresh material. This protocol was also used for the isolation of RNA from needles of spruce spp., from which 750-950 micrograms RNA per gram of starting material could routinely be obtained.

Base Sequence↗
PubMed2003

Isolation and purification of functional total RNA from different organs of cacao tree during its interaction with the pathogen Crinipellis perniciosa.

Witches' broom disease, caused by Crinipellis perniciosa, is one of the major fungal diseases causing severe losses to cacao tree (Theobroma cacao L.) plantations in South America. One of the challenges associated with the understanding of the cacao and Crinipellis interaction in genomic studies is the isolation of intact nucleic acids. In this report, we describe a new, successful, and reliable procedure for the isolation of RNA from tissues of cacao tree, both infected and uninfected by Crinipellis. This protocol overcomes the problems associated with the very high amount of polyphenols and polysaccharides present in cacao organs that are not easily removed by conventional extraction procedures. The protocol requires few reagents, uses ultracentrifugation and inexpensive consumables, and can be easily applied in any laboratory. This method produced high-quality RNA that was suitable for subsequent purposes, such as reverse transcription PCR and cDNA library construction. We also report the first evidence of RNA isolation from cacao organs infected by C. perniciosa such as meristems and fruits.

Basidiomycota↗