Muto Hisashi, Takaki Yoshihiro, Hirai Miho, Mino Sayaka, Sawayama Shigeki, Takai Ken, 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).
Microbes Environ. 2017 Dec 27;32(4):330-335. doi: 10.1264/jsme2.ME17048. Epub 2017 Nov 30.
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.
基于RNA的微生物分析，例如转录组分析和逆转录定量PCR，需要相对大量的高质量RNA。由于深海热液环境中微生物群落样本存在独特的矿物质且生物量较低，基于RNA的微生物群落分析在RNA提取方面常常遇到方法上的困难。在本研究中，我们评估了用于具有复杂矿物成分的深海热液喷口烟囱的RNA提取方法。使用模拟烟囱矿物和大肠杆菌总RNA溶液进行了矿物-RNA吸附实验，结果表明可检测到的RNA显著减少，这可能是由于RNA吸附到矿物上所致。添加三聚磷酸钠（STPP）、脱氧核苷酸三磷酸（dNTPs）、鲑鱼精DNA和氢氧化钠可防止RNA的这种减少。当使用TRIzol试剂和RNeasy柱时，添加STPP对从大肠杆菌细胞和模拟烟囱矿物的混合物中提取RNA也有效，但使用RNeasy PowerSoil总RNA试剂盒时则无效。STPP、TRIzol试剂、RNeasy柱和超声处理相结合，可从天然烟囱中获得最高的RNA产量。这种间接提取方法简单、快速、成本低，可用于大规模RNA提取。
