A Simple and Efficient RNA Extraction Method from Deep-Sea Hydrothermal Vent Chimney Structures

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  • Muto Hisashi
    Laboratory of Marine Environmental Microbiology, Graduate School of Agriculture, Kyoto University
  • Takaki Yoshihiro
    Department of Subsurface Geobiological Analysis and Research (D-SUGAR), Japan Agency for Marine-Earth Science and Technology (JAMSTEC)
  • Hirai Miho
    Research and Development (R&D) Center for Marine Biosciences, Marine Functional Biology Group (MFbio), Japan Agency for Marine-Earth Science and Technology (JAMSTEC)
  • Mino Sayaka
    Laboratory of Microbiology, Faculty of Fisheries Sciences, Hokkaido University
  • Sawayama Shigeki
    Laboratory of Marine Environmental Microbiology, Graduate School of Agriculture, Kyoto University
  • Takai Ken
    Department of Subsurface Geobiological Analysis and Research (D-SUGAR), Japan Agency for Marine-Earth Science and Technology (JAMSTEC)
  • Nakagawa Satoshi
    Laboratory of Marine Environmental Microbiology, Graduate School of Agriculture, Kyoto University Department of Subsurface Geobiological Analysis and Research (D-SUGAR), Japan Agency for Marine-Earth Science and Technology (JAMSTEC)

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<p>RNA-based microbiological analyses, e.g., transcriptome and reverse transcription-quantitative PCR, require a relatively large amount of high quality RNA. RNA-based analyses on microbial communities in deep-sea hydrothermal environments often encounter methodological difficulties with RNA extraction due to the presence of unique minerals in and the low biomass of samples. In the present study, we assessed RNA extraction methods for deep-sea vent chimneys that had complex mineral compositions. Mineral-RNA adsorption experiments were conducted using mock chimney minerals and Escherichia coli total RNA solution, and showed that detectable RNA significantly decreased possibly due to adsorption onto minerals. This decrease in RNA was prevented by the addition of sodium tripolyphosphate (STPP), deoxynucleotide triphosphates (dNTPs), salmon sperm DNA, and NaOH. The addition of STPP was also effective for RNA extraction from the mixture of E. coli cells and mock chimney minerals when TRIzol reagent and the RNeasy column were used, but not when the RNeasy PowerSoil total RNA kit was used. A combination of STPP, TRIzol reagent, the RNeasy column, and sonication resulted in the highest RNA yield from a natural chimney. This indirect extraction procedure is simple, rapid, inexpensive, and may be used for large-scale RNA extraction.</p>

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