Format

Send to

Choose Destination
Microbes Environ. 2017 Dec 27;32(4):330-335. doi: 10.1264/jsme2.ME17048. Epub 2017 Nov 30.

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

Author information

1
Laboratory of Marine Environmental Microbiology, Graduate School of Agriculture, Kyoto University.
2
Department of Subsurface Geobiological Analysis and Research (D-SUGAR), Japan Agency for Marine-Earth Science and Technology (JAMSTEC).
3
Research and Development (R&D) Center for Marine Biosciences, Marine Functional Biology Group (MFbio), Japan Agency for Marine-Earth Science and Technology (JAMSTEC).
4
Laboratory of Microbiology, Faculty of Fisheries Sciences, Hokkaido University.

Abstract

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.

KEYWORDS:

RNA extraction; chimney structure; deep-sea hydrothermal vent; microbial community

PMID:
29187693
PMCID:
PMC5745017
DOI:
10.1264/jsme2.ME17048
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for J-STAGE, Japan Science and Technology Information Aggregator, Electronic Icon for PubMed Central
Loading ...
Support Center