[Objective] RNA extraction is considered the key for soil metatranscriptomics and it is expected that different methods would have generated distinct resolution of active soil microbiome.However,it remains largely unknown about the quantitative bias assessment of soil microbial communities associated with commercial Kits RNA (KR) and Manual RNA extraction (MR) methods.The aim of this study was to assess the bias of metatranscriptomics associated with total RNA extractions by commercial kits and manual methods from three geographically distinct paddy soils.[Methods] Three kinds of paddy soils were collected,representing distinctly different origin of parent materials including viscous black sand in Hailun city of Heilongjiang province,sandy loam in Binhai city of Jiangsu province,quaternary red clay in Yingtan city of Jiangxi province.Total RNA was obtained by commercial kit method and manual methods.The quantity and quality of total RNA were assessed by ultraviolet spectrophotometry and agarose gel electrophoresis.The abundance and composition of soil microbiome was analyzed by real-time quantitative PCR and high-throughput sequencing of cDNA reversely transcribed from 16S rRNA.[Results] The purity of RNA extracted by kit method was higher than that of manual method,but it did not hold true for the quantity of RNA extracts.The kit method generated higher quantity of soil RNA extract from paddy soils with higher organic matters from Heilongjiang and Jiangsu provinces,while the manual MR method recovered more RNA from paddy soil in Yingtan city of Jiangxi province with lower organic matter.High-throughput sequencing revealed a total of 27 phyla and 409 genera in three paddy soils,and 19 phyla and 181 genera were statistically significantly biased,i.e.,the significant difference in relative abundance of these phylotypes was observed between manual MR and kit KR methods.These 19 phyla and 181 genera averaged for 40.4％ and 44.4％ of the total microbial abundance in soils.There are 11 phyla with higher relative abundance by KR than MR methods,and it is noteworthy that only Armatimonadetes phylum was KR-specifically biased,i.e.,it is consistently detected in three soils with higher abundance by KR than MR methods.Similarly,the MR method also led to higher abundance of 11 phyla than KR method,and the Firmicutes is the only phylum for MR-specific bias,i.e.,it is consistently observed with higher abundance in three soils by MR than KR method.At the genus level,the kit method consistently revealed two genera with higher relative abundance in all three paddy soils than manual method,and five genera were preferentially recovered by MR method in all three soils.We further assessed a total of 72 numerically dominant genera that could be recovered from all three soils having a relativeabundance＞0.1％.These genera accounted for more than 80％ of total microbial abundance in the three soils.The result suggested that 48 out of 72 genera have the same changing patterns of relative abundance among three paddy soils regardless of KR and MR methods.For example,the manual RNA extraction method indicated that the relative abundance of aerobic methanotrophs could be arranged in a decreasing order as Heilongjiang (1.68％)＞Jiangxi (0.90％)＞ Jiangsu (0.59％),while the kit KR RNA extraction method has the same order as Heilongjiang (0.52％)＞ Jiangxi (0.18％)＞ Jiangsu (0.13％).[Conclusion] Among 27 phyla and 409 genera detected in paddy soils,there are only 2 phyla and 7 genera that was consistently biased in all three paddy soils by either kits KR or manual MR methods.It suggests that the biased phylotypes associated with the RNA extraction method itself could be insignificant,accounting only for 7.4％ and 1.7％ of the total phyla and genera respectively.Although the quantity and quality of RNA extracted from paddy soil are obviously different between manual MR and kit KR methods,the bias associated with these two RNA extraction methods has no significant impact on the biogeographic patterns of soil microbiomes in the three paddy soil tested.It is estimated that 70％ of phyla and 22％of genera detected were observed with statistically significant difference between KR and MR methods.However,both RNA extraction methods could lead to the same conclusions regarding the changing patterns in relative abundance of microbial phylotpes among the three paddy soil tested.Despite the fact that the detection of every individual phylotype cannot be entirely reproduced by both methods and there are huge difference between these two methods,it seems plausible that the difference among soil types is sufficiently large so that the recovery of microbial communities would not be biased by RNA extraction method itself.These results imply that the method-specific bias of phylotype detection is much less than expected during soil RNA extraction.For future study the choice of RNA extraction method may not be of significant help,and the priority is to have experimental manipulation and treatments that would select for microbiomes with difference significantly larger than the bias associated with RNA extraction methods.%[目的]比较手工法和试剂盒法提取土壤RNA对原核微生物多样性的影响,评价两种方法研究土壤宏转录组的技术偏好性.[方法]针对黑龙江海伦砂质黑壤土、江苏滨海黏心夹砂土、江西鹰潭第四纪红黏土不同母质发育形成的三种水稻土,采用手工和试剂盒法分别获得微生物总RNA,利用高通量测序原核微生物16S rRNA多样性,通过紫外分光和琼脂糖凝胶电泳分析RNA质量,比较手工和试剂盒法提取RNA对水稻土原核微生物群落的影响规律.[结果]三种水稻土中试剂盒提取RNA的纯度皆高于手工法,但RNA提取总量不一致.试剂盒提取黑龙江和江苏水稻土的RNA总量高于手工法,而手工法提取的江西水稻土RNA总量高于试剂盒法.高通量测序发现土壤类型而不是RNA提取方法决定了原核微生物多样性指数和群落结构.3个水稻土共检测到27门和409属,两种RNA提取方法偏好性提取了19门和181属,这些门和属占水稻土微生物总丰度平均值分别为40.4％和44.4％.与手工提取RNA相比,试剂盒法发现11个微生物门的丰度显著偏高(P＜0.05),但仅有Armatimonadetes门在三种水稻土同时存在专一偏好性;手工法提取也发现1 1个微生物门的丰度显著高于试剂盒法,并且仅有Firmicutes门在三种水稻土中同时存在专一偏好性.在微生物属水平,三种水稻土中均发现试剂盒法偏好性提取了2属,而手工法偏好性提取了5属.进一步针对72个优势微生物属(丰度＞0.1％且同时存在于三种水稻土),发现其占所有微生物丰度80％强,且48属的变化规律与RNA提取方法无关.例如,手工法提取水稻土好氧甲烷氧化菌的规律为:黑龙江(1.68％)＞江西(0.90％)＞江苏(0.59％),而试剂盒法也得到一致结果,黑龙江(0.52％)＞江西(0.18％)＞江苏(0.13％).[结论]两种RNA提取方法本身的特异偏好性较小.三种水稻土27门409属中,仅有2门7属可能存在方法本身的专一偏好性,即在三种水稻土中,这些微生物均被试剂盒法或手工法特异性偏好提取,占所有原核微生物门和属比例仅为7％和1％左右.此外,针对同一水稻土,手工和试剂盒法提取RNA的总量、纯度、微生物相对丰度明显不同,在原核微生物门和属的分类水平,约70％的门和22％的属的丰度具有统计显著性差异,但两种RNA提取方法均能反映优势微生物类群在三种水稻土中的变异规律,土壤类型对原核微生物多样性的影响远大于RNA提取方法本身的偏好性.未来原核微生物宏转录组研究中,应优先考虑科学问题及其实验处理可能导致的微生物组及其转录差异,结合微生物RNA提取特点,最大限度地发挥宏转录组技术优势.
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