骆驼总RNA的提取方法选择及TRIzol与脾脏比例对RNA质量的影响  

Selection of camel total RNA extraction method and the effect of TRIzol to spleen ratio on RNA quality

作　　者：李全允 伊丽[1] 吉日木图[1,2] LI Quanyun;YI Li;JIRIMUTU(Key Laboratory of Dairy Biotechnology and Bioengineering,Ministry of Education,Inner Mongolia Agricultural University,Hohhot 010018,China;Camel Protection Association of Inner Mongolia,Badanjilin 737300,China)

机构地区：[1]内蒙古农业大学乳品生物技术与工程教育部重点实验室,呼和浩特010018 [2]内蒙古骆驼研究院,内蒙古巴丹吉林737300

出　　处：《黑龙江畜牧兽医》2020年第22期138-143,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：内蒙古自然科学基金项目(2018BS03017);内蒙古自治区科技创新引导项目(KCMS2018048);高层次人才引进科研启动项目(NDYB2018-48);双一流学科创新团队建设项目(NDSC2018-14);教育援外项目丝路1+1科研合作项目.

摘　　要：为了构建高质量的纳米抗体库,研究采用硅基膜法(柱纯化试剂盒法)和沉淀法分别提取骆驼外周血淋巴细胞和脾脏总RNA,通过超微量分光光度计、琼脂糖凝胶电泳及逆转录-聚合酶链反应(RT-PCR)检测,对提取结果进行比较分析,并探究了TRIzol试剂与脾脏用量比例对RNA提取质量的影响,以筛选驼源样本RNA提取方法。结果表明:沉淀法提取样本RNA的得率是硅基膜法的2倍以上,OD260/OD280值和OD260/OD230值也均处于正常范围内;硅基膜法所提取样本RNA的RT-PCR结果出现明亮的目的条带。而TRIzol的用量对脾脏组织RNA的提取质量不会有显著影响,仅对其得率有部分影响。说明用沉淀法提取样本RNA效果更好,而用硅基膜法提取的驼源RNA更适用于构建抗体文库。In order to construct a high-quality nano antibody library, the study used the silicon-based membrane method(Column purification kit method) and precipitation method to extract camel peripheral blood lymphocytes and spleen total RNA. The extraction results were compared and analyzed by ultramicro spectrophotometer, agarose gel electrophoresis and reverse transcription-polymerase chain reaction(RT-PCR) detection, and the influence of the ratio of TRIzol reagent and spleen dosage on the quality of RNA extraction was investigated to screen the RNA extraction method of camel source samples. The results showed that the yield of RNA extracted by precipitation method was more than twice that of the silicon-based membrane method, the values of OD260/OD280 and OD260/OD230 were also within the normal range. RT-PCR result of sample RNA extracted by the silicon-based membrane method showed a bright target band, while the amount of TRIzol had no significant effect on the extraction quality of RNA from spleen tissues, only had a partial impact on its yield. The results indicated that the precipitation method was more effective, and the camel-derived RNA extracted by silicon-based membrane method was more suitable for the construction of the antibody library.

关 键 词：骆驼 纳米抗体文库 核糖核酸 淋巴细胞 沉淀法 硅基膜法 脾脏 

分 类 号：R392[医药卫生—免疫学]