Modified method for the extraction of mRNA from paddy soils
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Published on: January 11, 2017
Hongling Qin1, Xiangbi Chen2, Yafang Tang2
1Key Laboratory of Agro-ecological Processes in Subtropical Regions, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, 410125, China. huniu@isa.ac.cn.
Objective:
Paddy soils contain relatively greater organic matter and water contents than other soils thereby limiting effective mRNA extraction. A modification of the conventional mRNA soil extraction method specific to paddy soils is described.
Results:
Two main modifications for co-extraction of DNA and RNA are: (1) addition of 20 % (w/v) sodium dodecyl sulphate to 10 % (w/v) hexadecyltrimethylammonium bromide extraction buffer, and (2) fresh soil, initially frozen at -80 °C, is immediately immersed in extraction buffer. The high-quality total RNA extracted can be directly used in downstream analyses without an additional step to remove humic acid. RNA purification was conducted to remove 5S rRNA, and the mRNA was enriched by selectively digesting rRNA. cDNA synthesised by reverse transcriptase was not contaminated by the reagents or genomic DNA.
Conclusion:
The modified method for mRNA extraction from paddy soil is suitable for analysing the expression of microbial genes from fresh paddy soil.
