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Simultaneous extraction from bacterioplankton of total RNA and DNA suitable for quantitative structure and function analyses.

ABSTRACT: 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.

SUBMITTER: Weinbauer MG

PROVIDER: S-EPMC123726 | biostudies-literature | 2002 Mar

REPOSITORIES: biostudies-literature

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Simultaneous extraction from bacterioplankton of total RNA and DNA suitable for quantitative structure and function analyses.

Weinbauer Markus G MG Fritz Ingo I Wenderoth Dirk F DF Höfle Manfred G MG

Applied and environmental microbiology 20020301 3

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 ...[more]

PMID: 11872453

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Project description:OBJECTIVES:Systematic reviews and meta-analyses are labor-intensive and time-consuming. Automated extraction of quantitative data from primary studies can accelerate this process. ClinicalTrials.gov, launched in 2000, is the world's largest trial repository of results data from clinical trials; it has been used as a source instead of journal articles. We have developed a Web application called EXACT (EXtracting Accurate efficacy and safety information from ClinicalTrials.gov) that allows users without advanced programming skills to automatically extract data from ClinicalTrials.gov in analysis-ready format. We have also used the automatically extracted data to examine the reproducibility of meta-analyses in three published systematic reviews. STUDY DESIGN AND SETTING:We developed a Python-based software application (EXACT) that automatically extracts data required for meta-analysis from the ClinicalTrials.gov database in a spreadsheet format. We confirmed the accuracy of the extracted data and then used those data to repeat meta-analyses in three published systematic reviews. To ensure that we used the same statistical methods and outcomes as the published systematic reviews, we repeated the meta-analyses using data manually extracted from the relevant journal articles. For the outcomes whose results we were able to reproduce using those journal article data, we examined the usability of ClinicalTrials.gov data. RESULTS:EXACT extracted data at ClincalTrials.gov with 100% accuracy, and it required 60% less time than the usual practice of manually extracting data from journal articles. We found that 87% of the data elements extracted using EXACT matched those extracted manually from the journal articles. We were able to reproduce 24 of 28 outcomes using the journal article data. Of these 24 outcomes, we were able to reproduce 83.3% of the published estimates using data at ClinicalTrials.gov. CONCLUSION:EXACT (http://bio-nlp.org/EXACT) automatically and accurately extracted data elements from ClinicalTrials.gov and thus reduced time in data extraction. The ClinicalTrials.gov data reproduced most meta-analysis results in our study, but this conclusion needs further validation.

Dataset Information

Simultaneous extraction from bacterioplankton of total RNA and DNA suitable for quantitative structure and function analyses.

Publications

Simultaneous extraction from bacterioplankton of total RNA and DNA suitable for quantitative structure and function analyses.

Similar Datasets

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