Abstract: A modified sodium dodecyl sulfate (SDS)-phenol method was proposed for total RNA extraction from fermented grains of Chinese strong-flavor Baijiu, a complex microecological environment unique to China. This method was compared with other common methods including commercial kit, Trizol method, hexadecyl trimethyl ammonium bromide (CTAB) method and sodium laurate method in regard to cost, extraction time, RNA concentration and purity, electrophoresis results, reverse transcription efficiency and high-throughput sequencing analysis. The results showed that the SDS-phenol and CTAB methods were inexpensive, while the kit method was the most time saving but most expensive. The total RNA concentration extracted by the kit method was the highest, which was 1.84, 2.33, 19.14 and 3.09 times as high as that by the modified SDS-phenol, Trizol, CTAB and sodium laurate methods, respectively. The OD260 nm/OD280 nm and OD260 nm/OD230 nm ratio of total RNA extracted by the SDS-phenol and kit methods were greater than 1.8 and 2.0, respectively. The electrophoresis showed more intact bands for total RNA extracted by the SDS-phenol method as compared to that extracted by the other methods, and but no bands for total RNA extracted the by the CTAB method. RNA extracted by the modified SDS-phenol, kit and Trizol methods could be efficiently reversely transcribed into cDNA and used for high-throughput sequencing analysis. The sequencing results showed that the dominant microbial genera (Lactobacillus and Clostridium) obtained by the three RNA extraction methods and the change of microbial population abundance in fermented grains at different fermentation times were basically the same. Among the three methods, the SDS-phenol method gave the highest diversity of microbial communities and could be useful to determine the differences in microbial groups among fermented grains at different fermentation times. Both the Trizol and kit methods could preferentially detect one more genus, but at very low levels (0.01%). In general, the modified SDS-phenol method was advantageous over the other RNA extraction methods. This study provides a theoretical basis for molecular biological research on fermented grains of Chinese Baijiu based on RNA in the future, and it is of reference significance for RNA extraction from environmental samples rich in sugar, humus and phenols.

Key words: fermented grains of Chinese strong-flavor Baijiu; metatranscriptome; total RNA extraction; evaluation of extraction methods; microbial community