title: Effect of DNA extraction and sample preservation method on rumen bacterial population.
abstract: 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.
authors.affiliation: Institute of Animal Physiology and Genetics, CAS, v.v.i., Vídeňská 1083, Prague 14220, Czech Republic. Electronic address: fliegerova@iapg.cas.cz.
authors.forename: Katerina
authors.initials: K
authors.lastname: Fliegerova
authors.affiliation: MTT Agrifood Research Finland, Animal Genomics, FI-31600 Jokioinen, Finland.
authors.forename: Ilma
authors.initials: I
authors.lastname: Tapio
authors.affiliation: Laboratoire d'Ecologie Alpine, CNRS, 2233 rue de la Piscine, 38041 Grenoble, France.
authors.forename: Aurelie
authors.initials: A
authors.lastname: Bonin
authors.affiliation: Institute of Animal Physiology and Genetics, CAS, v.v.i., Vídeňská 1083, Prague 14220, Czech Republic.
authors.forename: Jakub
authors.initials: J
authors.lastname: Mrazek
authors.affiliation: Università Cattolica del Sacro Cuore, Piacenza, Italy.
authors.forename: Maria Luisa
authors.initials: ML
authors.lastname: Callegari
authors.affiliation: Università Cattolica del Sacro Cuore, Piacenza, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Bani
authors.affiliation: MTT Agrifood Research Finland, Animal Nutrition and Nutrigenomics, FI-31600 Jokioinen, Finland.
authors.forename: Alireza
authors.initials: A
authors.lastname: Bayat
authors.affiliation: MTT Agrifood Research Finland, Animal Genomics, FI-31600 Jokioinen, Finland.
authors.forename: Johanna
authors.initials: J
authors.lastname: Vilkki
authors.affiliation: Institute of Animal Physiology and Genetics, CAS, v.v.i., Vídeňská 1083, Prague 14220, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Kopečný
authors.affiliation: MTT Agrifood Research Finland, Animal Nutrition and Nutrigenomics, FI-31600 Jokioinen, Finland.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Shingfield
authors.affiliation: Laboratoire d'Ecologie Alpine, CNRS, 2233 rue de la Piscine, 38041 Grenoble, France.
authors.forename: Frederic
authors.initials: F
authors.lastname: Boyer
authors.affiliation: Laboratoire d'Ecologie Alpine, CNRS, 2233 rue de la Piscine, 38041 Grenoble, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Coissac
authors.affiliation: Laboratoire d'Ecologie Alpine, CNRS, 2233 rue de la Piscine, 38041 Grenoble, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Taberlet
authors.affiliation: Rowett Institute of Nutrition and Health, University of Aberdeen, Bucksburn, Aberdeen AB21 9SB, United Kingdom.
authors.forename: R John
authors.initials: RJ
authors.lastname: Wallace
mesh_terms: D000818:Animals; D041963:Bacteroidetes / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D002417:Cattle; D015925:Cryopreservation; D058893:DNA Barcoding, Taxonomic; D004269:DNA, Bacterial / Q000302:isolation & purification*; D058645:Denaturing Gradient Gel Electrophoresis; D006094:Gram-Positive Bacteria / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D059014:High-Throughput Nucleotide Sequencing; D010802:Phylogeny; D060888:Real-Time Polymerase Chain Reaction; D012417:Rumen / Q000382:microbiology*; D013048:Specimen Handling / Q000379:methods*
chemical_list: D004269:DNA, Bacterial
keywords: Bacterial diversity; Bacteroidetes; Extracellular DNA; Firmicutes; Intracellular DNA; Metabarcoding; PCR-DGGE; Q-PCR; Rumen fluid; Storage conditions