Effect of DNA extraction and sample preservation method on rumen bacterial population
Language English Country England, Great Britain Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
24125910
DOI
10.1016/j.anaerobe.2013.09.015
PII: S1075-9964(13)00167-4
Knihovny.cz E-resources
- Keywords
- Bacterial diversity, Bacteroidetes, Extracellular DNA, Firmicutes, Intracellular DNA, Metabarcoding, PCR-DGGE, Q-PCR, Rumen fluid, Storage conditions,
- MeSH
- Rumen microbiology MeSH
- Bacteroidetes classification genetics isolation & purification MeSH
- Denaturing Gradient Gel Electrophoresis MeSH
- DNA, Bacterial isolation & purification MeSH
- Phylogeny MeSH
- Gram-Positive Bacteria classification genetics isolation & purification MeSH
- Cryopreservation * MeSH
- Real-Time Polymerase Chain Reaction MeSH
- Specimen Handling methods MeSH
- Cattle MeSH
- DNA Barcoding, Taxonomic * MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Animals MeSH
- Check Tag
- Cattle MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA, Bacterial MeSH
The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, -80 °C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P < 0.01) in eDNA samples. The qPCR indicated significantly higher amount of Firmicutes in iDNA sample frozen with glycerol (P < 0.01). Deep sequencing analysis of iDNA samples revealed the prevalence of Bacteroidetes and similarity of samples frozen with and without cryoprotectants, which differed from sample stored with ethanol at room temperature. Centrifugation and consequent filtration of rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at -80 °C was found as the optimal method to study ruminal bacterial profile.
Institute of Animal Physiology and Genetics CAS v v i Vídeňská 1083 Prague 14220 Czech Republic
Laboratoire d'Ecologie Alpine CNRS 2233 rue de la Piscine 38041 Grenoble France
MTT Agrifood Research Finland Animal Genomics FI 31600 Jokioinen Finland
MTT Agrifood Research Finland Animal Nutrition and Nutrigenomics FI 31600 Jokioinen Finland
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