[1]吴思琪,宋静颐,秦倩,等.一种高效稳定的微生物总RNA提取方法[J].江苏农业学报,2017,(03):517-523.[doi:doi:10.3969/j.issn.1000-4440.2017.03.006]
　WU Si-qi,SONG Jing-yi,QIN Qian,et al.A high-efficiency and stable method for extracting total RNA from microorganisms[J].,2017,(03):517-523.[doi:doi:10.3969/j.issn.1000-4440.2017.03.006]
点击复制

一种高效稳定的微生物总RNA提取方法()
分享到：

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2017年03期
页码:
517-523
栏目:
遗传育种·生理生化
出版日期:
2017-06-30

文章信息/Info

Title:
A high-efficiency and stable method for extracting total RNA from microorganisms
作者:
吴思琪1; 宋静颐1; 秦倩2; 张策3; 金君华1; 谢远红1; 刘慧1; 张红星1
（1.北京农学院食品科学与工程学院食品质量与安全北京实验室/农产品有害微生物及农残安全检测与控制北京市重点实验室/微生态制剂关键技术开发北京市工程实验室/北京市食品安全免疫快速检测工程技术研究中心，北京102206；2.中国青年政治学院，北京100089；3.北京市经济管理学校，北京100142）
Author(s):
WU Si-qi1; SONG Jing-yi1; QIN Qian2; ZHANG Ce3; JIN Jun-hua1; XIE Yuan-hong1; LIU Hui1; ZHANG Hong-xing1
（1.Food Science and Engineering College, Beijing Laboratory of Food Quality and Safety/ Beijing Key Laboratory of Detection and Control of Spoilage Organisms and Pesticide Residues in Agricultural Products/ Beijing Engineering Laboratory of Probiotics Key Technology Development/ Beijing Engineering Technology Research Center of Food Safety Immune Rapid Detection, Beijing 102206, China;2.China Youth University for Political Sciences, Beijing 100089, China;3.Beijing Economic Management School, Beijing 100142, China）
关键词:
微生物; RNA提取; 玻璃珠-酚仿
Keywords:
microorganism; RNA extraction; glass bead-phenol/chloroform
分类号:
Q522
DOI:
doi:10.3969/j.issn.1000-4440.2017.03.006
文献标志码:
A
摘要:
本研究通过对玻璃珠-酚仿RNA提取方法进行改进优化，实现对革兰氏阳性菌、革兰氏阴性菌和真菌这3类微生物RNA高效稳定的提取。结果表明，玻璃珠-酚仿提取法能有效提取的RNA最低菌体细胞量为 1×107个，远低于试验所选商业化试剂盒所需细胞量。以 1×107个细胞量为例，利用Beadbeater仪破碎30 s，使用直径100 μm的玻璃珠破碎提取得到的细菌RNA浓度显著高于使用直径500 μm的玻璃珠提取的。使用2种直径的玻璃珠所得酵母菌RNA浓度无显著性差异。在直径100 μm的条件下，破碎30 s即可保证RNA的高效提取，对于长双歧杆菌、大肠杆菌和酵母菌，破碎时间长达150 s也不会引起RNA降解。本法所提RNA的A260/A280为1.96～210，A260/A230为 2.03～244，浓度和纯度均优于试验所选商业化试剂盒所提的RNA。
Abstract:
In this study, the glass bead-phenol/chloroform RNA extraction method was optimized to realize the highly efficient and stable extraction of RNA from three kinds of microorganisms， gram positive bacteria, gram negative bacteria and fungus. The minimum thalli cell number that could be extracted by glass bead-phenol RNA extraction was 1×107, far less than that extracted by commercialized kit. The concentration of bacterial RNA extracted by glass beads with Φ= 100 μm from 1×107 cells was significantly higher than that with Φ= 500 μm. For the yeast, there was no significant difference in RNA concentration extracted by glass beads with different diameters. Glass beads with 100 μm diameter breaking for 30 s could ensure the efficient extraction of RNA, and the degradation of RNA did not happen even broken for 150 s for Bifidobacterium longum, Escherichia coli and yeast. The concentration and purity of the RNA extracted by optimized method were better than those by commercialized kit.

参考文献/References:

[1]LANDOLT L, MARTI H P, BEISLAND C, et al. RNA extraction for RNA sequencing of archival renal tissues[J]. Scandinavian Journal of Clinical and Laboratory Investigation, 2016, 76(5): 426-434.
[2]刘波，张晓明，郭巧生，等．百蕊草根系总RNA提取方法比较及优化［J］．江苏农业科学，2015,43(1):44-46.
[3]张春兰,秦孜娟,王桂芝,等. 转录组与RNA-Seq技术[J]. 生物技术通报, 2012(12): 51-56.
[4]LI J H, TANG C H, SONG C Y, et al. A simple, rapid and effective method for total RNA extraction from Lentinula edodes[J]. Biotechnology Letters, 2006, 28(15): 1193-1197.
[5]尹珍珍,吴光斌,陈发河,等. 莲雾果实高质量总RNA提取方法的建立[J]. 食品科学, 2015(14):1-4.
[6]胡朝辉,安家彦,高慧利. 质壁分离剂盐法提取啤酒酵母RNA[J]. 煤炭与化工, 2012, 35(1):33-35.
[7]ZHU J, ZHAO L, GUO H Y, et al. Immunomodulatory effects of novel bifidobacterium and lactobacillus strains on murine macrophage cells[J]. African Journal of Microbiology Research, 2011, 5(1): 8-15.
[8]王海珍,王加启,黄庆生. 乳酸菌类微生物制剂的作用机理及其应用[J]. 养殖与饲料, 2005(3):9-11.
[9]LIDBECK A, VERVIK E, RAFTER J, et al. Effect of Lactobacillus acidophilus supplements on mutagen excretion in faeces and urine in humans[J]. Microbial Ecology in Health and Disease, 1992, 5(1): 59-67.
[10]解庭波. 大肠杆菌表达系统的研究进展[J]. 长江大学学报(自然科学版), 2008, 5(3): 77-82.
[11]祁浩,刘新利. 大肠杆菌表达系统和酵母表达系统的研究进展[J]. 安徽农业科学, 2016, 44(17): 4-6.
[12]KOLIJN K, LEENDERS G J L H. Comparison of RNA extraction kits and histological stains for laser capture microdissected prostate tissue[J]. BMC Research Notes, 2016, 9: 17.
[13]陈星,潘迎捷,孙晓红,等. 细菌总RNA提取方法的研究进展[J]. 湖南农业科学, 2010, 2010(5): 9-11.
[14]罗绍银,高继海,叶生亮,等. 一种提取富含油脂植物组织总RNA的改良SDS酸酚法[J]. 应用与环境生物学报, 2009, 15(2): 284-287.
[15]LIAO Z H, CHEN M, GUO L, et al. Rapid isolation of high-quality total RNA from Taxus and Ginkgo[J]. Preparative Biochemistry and Biotechnology, 2004, 34(3): 209-214.
[16]申艳红,陈晓静. 改良热硼酸小量法提取番木瓜果肉总RNA[J]. 中国南方果树, 2009, 38(2): 7-9.
[17]TAN S C, YIAP B C. DNA, RNA, and protein extraction: the past and the present[J]. BioMed Research International, 2009, 2009:1-10.
[18]于寒松,彭帅,谢远红,等. 一种RNA提取试剂盒——TRIZOL的使用方法初探[J]. 食品科学, 2005, 26(11): 39-42.
[19]VANDECASTEELE S J, PEETERMANS W E, MERCKX R, et al. Use of gDNA as internal standard for gene expression in staphylococci in vitro and in vivo[J]. Biochemical & Biophysical Research Communications, 2002, 291(3): 528-534.
[20]YE W, LIU L, ZHENG W, et al. Comparison of RNA extraction methods applied to gene cloning of the taxol-producing fungi[J]. African Journal of Microbiology Research, 2009, 3(10):632-636.
[21]TRIANT D A, WHITEHEAD A. Simultaneous extraction of high-quality RNA and DNA from small tissue samples[J]. Journal of Heredity, 2009, 100(2): 246-250.
[22]易弋,容元平,程谦伟,等. 不同破壁方法提取酵母菌总RNA的比较[J]. 食品科学, 2011, 32(11): 161-164.
[23]王佳,易弋,杨军,等. 不同破壁方法提取无乳链球菌质粒DNA的比较[J]. 食品科技, 2013(5): 22-26.
[24]戈海泽,郭刚,张瑞,等. 玻璃珠法提取基因组DNA[J]. 天津医科大学学报, 2006, 12(2): 313-314.
[25]邹晓蕾,刘礼崔,罗立新. 细菌总RNA提取方法的比较[J]. 现代食品科技, 2013(8): 1948-1954.
[26]张仕林，许玉超，帅强，等．洋葱不同组织RNA提取方法比较分析［J］．江苏农业科学，2016,44(10):95-97.
[27]GANCZ D, GILBOA L. RNA isolation from early drosophila larval ovaries[J]. Germline Stem Cells, 2017,1463:75-83.
[28]郝雪英，丰震，吕传青. ‘鲁红一号’元宝枫叶片总RNA提取方法的比较研究[J]. 山东农业科学,2015,47(3):5-8.
[29]宗成志,左欣欣,严海燕. 影响RNA提取质量的因素分析[J]. 广西农学报, 2011, 26(1): 31-34.
[30]KAUZNA M, KURAS A, MIKICISKI A, et al. Evaluation of different RNA extraction methods for high-quality total RNA and mRNA from Erwinia amylovora in planta[J]. European Journal of Plant Pathology, 2016, 146(4): 893-899.
[31]刘朝,乔建军,朱宏吉. 乳酸菌肽聚糖的研究进展[J]. 微生物学通报, 2016, 43(1): 188-197.
[32]沈萍,陈向东. 微生物学[M]. 8版.北京：高等教育出版社，2016:33-34.

相似文献/References:

[1]王廷峰,赵密珍,关玲,等.玉米套作及秸秆还田对草莓连作土壤养分及微生物区系的影响[J].江苏农业学报,2019,(06):1421.[doi:doi:10.3969/j.issn.1000-4440.2019.06.022]
　WANG Ting-feng,ZHAO Mi-zhen,GUAN-Ling,et al.Effects of intercropping with corn and straw returning on nutrients and microflora in strawberry continuous cropping soil[J].,2019,(03):1421.[doi:doi:10.3969/j.issn.1000-4440.2019.06.022]
[2]龚云辉,刘云根,杨思林,等.pH值对高原山地分散养殖型农村沟渠底泥磷释放的影响[J].江苏农业学报,2020,(04):955.[doi:doi:10.3969/j.issn.1000-4440.2020.04.021]
　GONG Yun-hui,LIU Yun-gen,YANG Si-lin,et al.Effects of pH value on the release of phosphorus from the sediment in scattered farming rural ditch in plateau region[J].,2020,(03):955.[doi:doi:10.3969/j.issn.1000-4440.2020.04.021]
[3]杨雍康,药栋,李博,等.微生物群落在修复重金属污染土壤过程中的作用[J].江苏农业学报,2020,(05):1322.[doi:doi:10.3969/j.issn.1000-4440.2020.05.032]
　YANG Yong-kang,YAO Dong,LI Bo,et al.Effect of microbial community in the process of remediation of heavy metal pollution in soil[J].,2020,(03):1322.[doi:doi:10.3969/j.issn.1000-4440.2020.05.032]
[4]罗志斌,欧慧敏,李建中,等.体外发酵法评估呼伦贝尔羊瘤胃对过瘤胃蛋氨酸的生物利用率及过瘤胃蛋氨酸对瘤胃微生物的影响[J].江苏农业学报,2024,(05):874.[doi:doi:10.3969/j.issn.1000-4440.2024.05.012]
　LUO Zhibin,OU Huimin,LI Jianzhong,et al.Evaluation for bioavailability of rumen protected methionine and its effects on rumen microorganisms in Hulun Buir sheep by in vitro fermentation[J].,2024,(03):874.[doi:doi:10.3969/j.issn.1000-4440.2024.05.012]

备注/Memo

备注/Memo:
收稿日期：2016-03-06 基金项目：北京市教委科研计划支持项目（KM201610020015）；北京市属高等学校高层次人才引进与培养长城学者计划（CIT&TGD20140315）;杨胜先生门生社群项目（C2016028） 作者简介：吴思琪（1994-），女，黑龙江牡丹江人，硕士研究生，主要从事食品生物技术研究。（Tel）15010295516；（E-mail）siqiwu0127@163.com。宋静颐为共同第一作者。通讯作者：张红星,（Tel）13810688219; (E-mail) hxzhang511@163.com
更新日期/Last Update: 2017-06-29