瘤胃微生物RNA提取中样品前处理方法的优化  

Optimization of Sample Pretreatment in Rumen Microbial RNA Extraction

作　　者：刘思佳 赵圣国[1] 郑楠[1] 王加启[1] LIU Si-jia;ZHAO Sheng-guo;ZHENG Nan;WANG Jia-qi(State Key Lab.of Anim.Nutrit′n,Inst.of Anim.Sci.,Chinese Acad.of Agric.Sci.,Beijing 100193)

机构地区：[1]中国农业科学院北京畜牧兽医研究所动物营养国家重点实验室,北京100193

出　　处：《微生物学杂志》2020年第1期88-93,共6页Journal of Microbiology

基　　金：国家重点研发计划项目(2017YFD0500502);国家自然科学基金项目(31430081);中国农业科学院农业科技创新工程重大产出科研选题项目(CAAS-ZDXT2019004);中国农业科学院科技创新工程项目(ASTIP-IAS12)。

摘　　要：通过优化瘤胃液前处理方法,提高瘤胃微生物RNA提取数量与质量。试验优化4个前处理条件,共组合形成5个处理组。前处理完成后,利用Trizol进行RNA提取,检测RNA浓度和完整度(RIN)。结果显示,提取瘤胃内容物和瘤胃菌体RNA浓度和完整度的影响没有显著差异(P>0.05)。液氮保存的样品提取的RNA浓度和完整度均显著(P<0.05)高于RNA保护液保存的样品。解冻处理对RNA浓度的影响无显著差异(P>0.05),但是不解冻处理提取的RNA完整度显著高于解冻处理(P<0.05)。液氮冷冻研磨提取的RNA浓度(P<0.05)显著高于常温研磨,但是对RNA完整度的影响没有显著差异(P>0.05)。瘤胃内容物、液氮速冻、不解冻处理结合冷冻研磨方法所提取RNA的RIN值为9.43,完整度最高。瘤胃内容物、液氮速冻、解冻离心收集菌体结合冷冻研磨方法所提取的RNA浓度为1048 ng/μL,为最高浓度。因此,瘤胃内容物直接液氮速冻并在不解冻条件下冷冻研磨,所提取的RNA效果最好,可用于后续宏转录组学试验。The aim of this study was to optimize the pretreatment of rumen fluid and improve the quantity and quality of RNA extraction in rumen microbes.Four pretreatment conditions in the experiment were optimized,and the total combinations to form five treatment groups.After the pretreatment completed,RNA was extracted with Trizol,and detected the RNA concentration and integrity(RIN).The results showed that the concentration and integrity of RNA extracted from rumen contents and rumen microbial cells had no significant difference(P>0.05).The concentration and integrity of RNA extracted from samples preserved and ground in liquid nitrogen were significantly higher than those preserved in RNA protection solution(P<0.05).There was no significant difference in the effect of thawing treatment on RNA concentration(P>0.05),however the integrity of RNA extracted by rumen fluid with unthawing treatment was significantly higher than thawing treatment(P<0.05).The concentration of RNA extracted in liquid nitrogen frozen and ground was significantly higher(P<0.05)than that ground in room temperature,but there was no significant difference in the effect of RNA integrity(P>0.05).The RIN value of RNA extracted from rumen contents in liquid nitrogen quick-freezing and unthawed combined with freeze-grinding method was 9.43,the integrity was the highest.The concentration of RNA extracted from rumen contents using liquid nitrogen quickfreezing,thawed and centrifuged to collect microbial cells combined with freeze-grinding method was 1048 ng/μL,which was the highest concentration.Therefore,direct liquid nitrogen quick-freezing and grinding under unthawing condition,the rumen contents results in the best RNA extraction,and can be used in subsequent macro-transcriptome experiments.

关 键 词：瘤胃液 前处理 破碎温度 RNA提取 优化 RNA质量 

分 类 号：Q939.94[生物学—微生物学]