title: PCR Conditions for 16S Primers for Analysis of Microbes in the Colon of Rats.
abstract: The study of the composition of the intestinal flora is important to the health of the host, playing a key role in maintaining intestinal homeostasis and the evolution of the immune system. For these studies, various universal primers of the 16S rDNA gene are used in microbial taxonomy. Here, we report an evaluation of 5 universal primers to explore the presence of microbial DNA in colon biopsies preserved in RNAlater solution. The DNA extracted was used for the amplification of PCR products containing the variable (V) regions of the microbial 16S rDNA gene. The PCR products were studied by restriction fragment length polymorphism (RFLP) analysis and DNA sequence, whose percent of homology with microbial sequences reported in GenBank was verified using bioinformatics tools. The presence of microbes in the colon of rats was quantified by the quantitative PCR (qPCR) technique. We obtained microbial DNA from rat, useful for PCR analysis with the universal primers for the bacteria 16S rDNA. The sequences of PCR products obtained from a colon biopsy of the animal showed homology with the classes bacilli (Lactobacillus spp) and proteobacteria, normally represented in the colon of rats. The proposed methodology allowed the attainment of DNA of bacteria with the quality and integrity for use in qPCR, sequencing, and PCR-RFLP analysis. The selected universal primers provided knowledge of the abundance of microorganisms and the formation of a preliminary test of bacterial diversity in rat colon biopsies.
authors.affiliation: Pharmacogenomics Groups, Systems Biology Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors.forename: I A
authors.initials: IA
authors.lastname: Guillen
authors.affiliation: Pharmacogenomics Groups, Systems Biology Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors.forename: H
authors.initials: H
authors.lastname: Camacho
authors.affiliation: Pharmacogenomics Groups, Systems Biology Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors.forename: A D
authors.initials: AD
authors.lastname: Tuero
authors.affiliation: Pharmacogenomics Groups, Systems Biology Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors.forename: D
authors.initials: D
authors.lastname: Bacardí
authors.affiliation: Pharmacogenomics Groups, Systems Biology Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors.forename: D O
authors.initials: DO
authors.lastname: Palenzuela
authors.affiliation: Pharmacogenomics Groups, Systems Biology Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors.forename: A
authors.initials: A
authors.lastname: Aguilera
authors.affiliation: Pharmacogenomics Groups, Systems Biology Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors.forename: J A
authors.initials: JA
authors.lastname: Silva
authors.affiliation: Pharmacogenomics Groups, Systems Biology Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors.forename: R
authors.initials: R
authors.lastname: Estrada
authors.affiliation: Pharmacogenomics Groups, Systems Biology Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors.forename: O
authors.initials: O
authors.lastname: Gell
authors.affiliation: Pharmacogenomics Groups, Systems Biology Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors.forename: J
authors.initials: J
authors.lastname: Suárez
authors.affiliation: Pharmacogenomics Groups, Systems Biology Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors.forename: J
authors.initials: J
authors.lastname: Ancizar
authors.affiliation: Pharmacogenomics Groups, Systems Biology Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors.forename: E
authors.initials: E
authors.lastname: Brown
authors.affiliation: Pharmacogenomics Groups, Systems Biology Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors.forename: A B
authors.initials: AB
authors.lastname: Colarte
authors.affiliation: Pharmacogenomics Groups, Systems Biology Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors.forename: J
authors.initials: J
authors.lastname: Castro
authors.affiliation: Pharmacogenomics Groups, Systems Biology Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors.forename: L I
authors.initials: LI
authors.lastname: Novoa
mesh_terms: D000818:Animals; D001419:Bacteria / Q000235:genetics; D003106:Colon / Q000382:microbiology*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D000069196:Gastrointestinal Microbiome; D005798:Genes, Bacterial; D008297:Male; D058889:Molecular Typing / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D051381:Rats; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: RFLP; bacterial; biopsy; rDNA gene