AIM: Development of a method for the isolation and purification of metagenomic RNA (mgRNA) from the ectopic bacterial flora of octopus. METHODS AND RESULTS: Modifications were made to the methods of Valenzuela-Avendaño et al. (Plant Mol Biol Rep, 2005, 23, 199a) and Chomczynski and Sacchi (Anal Biochem, 1987, 162, 156) to develop a protocol based on chemical lysis with Trizol. This proposed protocol effectively isolated mgRNA. The resulting bacterial RNA transcripts were amplified with universal primers directed to the hypervariable regions of the 16S rRNA gene by complementary DNA synthesis. Protocol efficacy in the study of metabolically active bacterial flora was proven using DGGE, which produced a banding pattern that recovered sequences mainly related to the Vibrionaceae family. CONCLUSION: The analysed samples were clearly complex, and the proposed protocol was proven to effectively isolate mgRNA from the metabolically active bacterial flora associated with octopus. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first protocol proposed for the isolation of bacterial mgRNA that allows identification and study of metabolically active bacterial flora associated with octopus. This is an important step forward in understanding and controlling the microbial community of this economically important fishery resource, aimed at detecting its potentially pathogenic bacteria.
AIM: Development of a method for the isolation and purification of metagenomic RNA (mgRNA) from the ectopic bacterial flora of octopus. METHODS AND RESULTS: Modifications were made to the methods of Valenzuela-Avendaño et al. (Plant Mol Biol Rep, 2005, 23, 199a) and Chomczynski and Sacchi (Anal Biochem, 1987, 162, 156) to develop a protocol based on chemical lysis with Trizol. This proposed protocol effectively isolated mgRNA. The resulting bacterial RNA transcripts were amplified with universal primers directed to the hypervariable regions of the 16S rRNA gene by complementary DNA synthesis. Protocol efficacy in the study of metabolically active bacterial flora was proven using DGGE, which produced a banding pattern that recovered sequences mainly related to the Vibrionaceae family. CONCLUSION: The analysed samples were clearly complex, and the proposed protocol was proven to effectively isolate mgRNA from the metabolically active bacterial flora associated with octopus. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first protocol proposed for the isolation of bacterial mgRNA that allows identification and study of metabolically active bacterial flora associated with octopus. This is an important step forward in understanding and controlling the microbial community of this economically important fishery resource, aimed at detecting its potentially pathogenic bacteria.