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乙型肝炎病毒SYBR GreenⅠ实时荧光定量PCR方法的建立及临床应用 被引量:6
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作者 何维娜 吕东月 +3 位作者 刘和录 韩杰辉 何玥 李培培 《现代检验医学杂志》 CAS 2016年第3期98-101,共4页
目的建立 SYBR Green I实时荧光定量 PCR检测乙型肝炎病毒 DNA的快速方法,并探讨其临床应用价值。方法根据GenBank公布的Hepatitis B virus gp1基因序列设计引物,建立SYBR Green I实时荧光定量PCR,并对反应体系和扩增程序进行优化。... 目的建立 SYBR Green I实时荧光定量 PCR检测乙型肝炎病毒 DNA的快速方法,并探讨其临床应用价值。方法根据GenBank公布的Hepatitis B virus gp1基因序列设计引物,建立SYBR Green I实时荧光定量PCR,并对反应体系和扩增程序进行优化。定量标准品通过基因克隆方法获得,同时以浙江省夸克公司 HBV DNA定量检测试剂盒作对照,应用于随机选取的100份乙型肝炎患者血清检测。结果 SYBR Green I 实时荧光定量 PCR 检出限范围为5&#215;102 copies/ml~5&#215;108 copies/ml,HBV DNA浓度与CT值有良好线性关系,无交叉反应,整个过程仅需2.5 h。在随机抽取的100份临床标本应用中,与浙江省夸克公司的 HBV荧光定量 PCR检测试剂相比,建立的 SYBR Green I实时荧光定量PCR体系灵敏度100%,特异度92.5%。结论 SYBR Green实时荧光定量PCR快速、简便、灵敏度高、特异度强,可用于乙型肝炎患者病情监测,有效指导临床用药,准确评价 HBV感染者的病情。 展开更多
关键词 SYBR Green I实时荧光定量pcr 乙型肝炎病毒DNA 快速检测 公司产品对比
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实时荧光定量PCR检测皱纹盘鲍Δ5脂肪酸去饱和酶基因表达方法的建立及应用 被引量:2
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作者 李明珠 麦康森 +3 位作者 何艮 艾庆辉 徐玮 张文兵 《水生生物学报》 CAS CSCD 北大核心 2014年第2期328-334,共7页
研究以皱纹盘鲍(Haliotis discus hannai Ino)体内存在的2条Δ5 Fad(Hdhfad1和Hdhfad2)为目的基因,β-肌动蛋白(β-actin)和核糖体蛋白S9(Ribosomal protein S9,RPS9)为内参基因,应用2–ΔΔCt方法建立了实时荧光定量PCR(Real-Time quan... 研究以皱纹盘鲍(Haliotis discus hannai Ino)体内存在的2条Δ5 Fad(Hdhfad1和Hdhfad2)为目的基因,β-肌动蛋白(β-actin)和核糖体蛋白S9(Ribosomal protein S9,RPS9)为内参基因,应用2–ΔΔCt方法建立了实时荧光定量PCR(Real-Time quantitative PCR,RT-qPCR)检测体系,并应用此体系分析了鲍鱼肌肉组织Δ5 Fad在不同饲料处理下的表达差异。实验饲料含有不同的脂肪源,分别是棕榈酸甘油酯(Tripalmitin,TP饲料)、富含二十碳四烯酸(Arachidonic acid,ARA)的油脂(AO饲料)和富含二十碳五烯酸(Eicosapentaenoic acid,EPA)的油脂(EO饲料)。结果表明:针对Hdhfad1、Hdhfad2、β-actin和RPS9所设计的引物特异性强。各引物对的PCR扩增效率(Efficiency,E)分别为1.05、0.99、0.97和0.98,满足2–ΔΔCt方法对E的要求。当退火温度为52℃,反应体积为25μL时,RT-qPCR的扩增效果最好。所建立的体系能够准确定量Δ5 Fad的基因表达。利用该方法分析Δ5 Fad在不同饲料处理下的表达结果显示,与TP对照组相比,EO和AO饲料显著降低了鲍鱼肌肉组织Δ5 Fad(Hdhfad1和Hdhfad2)的表达量。 展开更多
关键词 脂肪酸去饱和酶 鲍鱼 基因表达
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鹿布鲁氏菌实时荧光定量PCR检测方法的建立 被引量:3
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作者 郝俊伟 张云 +6 位作者 杨宇航 刘红娜 王文玉 张秀丽 时坤 李建明 杜锐 《中国动物检疫》 CAS 2014年第2期71-76,共6页
本研究依据布鲁氏菌的特异性基因Omp25c的部分片段作为靶基因,设计探针引物,且优化了反应体系,筛选出引物、探针的最优浓度配比。将扩增产物连接到PGEM-T载体上,制备标准品及标准曲线,建立鹿布鲁氏菌荧光定量PCR检测方法,并对其特异性... 本研究依据布鲁氏菌的特异性基因Omp25c的部分片段作为靶基因,设计探针引物,且优化了反应体系,筛选出引物、探针的最优浓度配比。将扩增产物连接到PGEM-T载体上,制备标准品及标准曲线,建立鹿布鲁氏菌荧光定量PCR检测方法,并对其特异性、稳定性、敏感性进行评价。由标准曲线可知该方法的最低检测浓度可达到36拷贝/μL,比常规PCR灵敏度高出很多。 展开更多
关键词 鹿 布鲁氏菌
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流感病毒分型与亚型实时荧光定量PCR检测能力验证结果分析 被引量:1
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作者 魏明琳 《临床检验杂志(电子版)》 2017年第2期393-394,共2页
目的验证本实验室对流感病毒分型能力,以及实时荧光定量PCR检测能力,提高本实验室的管理水平以及技术人员的检测水平。方法组建检测小组,为本实验室检测人员发放制备样品,对流感病毒进行分型,采用实时荧光定量PCR进行检测,分析比较检测... 目的验证本实验室对流感病毒分型能力,以及实时荧光定量PCR检测能力,提高本实验室的管理水平以及技术人员的检测水平。方法组建检测小组,为本实验室检测人员发放制备样品,对流感病毒进行分型,采用实时荧光定量PCR进行检测,分析比较检测的结果。结果经实时荧光定量PCR检测,抽取所有样品均匀性和稳定性都比较好(P>0.05)。本实验室发放的总样品数是384个,经检测后结果与指定判定结果相同的样品有350个,合格率为91.14%。结论本实验室对流感病毒分型能力,以及亚型实时荧光定量PCR检测能力都比较好,相关技术人员的检测水平达到标准,能够为有关部门分析和预防流感提供有力的依据。 展开更多
关键词 流感病毒 时荧光定量pcr 检测能力
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实时荧光定量PCR在食品致病菌及转基因成分检测中应用 被引量:1
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作者 卢付青 唐善虎 +2 位作者 白菊红 闫利国 水旭亭 《粮食与油脂》 北大核心 2014年第12期16-19,共4页
与传统定性PCR技术相比,实时荧光定量PCR有更多的优点,其速度快,特异性更强、灵敏度更高、无污染性、自动化水平高等,且能对DNA模板进行定量。本文综述了实时荧光定量PCR技术原理、分类及其应用,并对其存在的问题和发展前景进行了展望。
关键词 致病菌 转基因成分 原理 应用
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实时荧光定量PCR检测腮腺沃辛瘤中Gadd45β表达 被引量:2
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作者 徐燕 郑建金 +3 位作者 李凤梅 李涛 董刚 于珊珊 《医学分子生物学杂志》 CAS 2016年第3期153-157,共5页
目的:从分子学水平检测腮腺沃辛瘤相对于瘤旁腮腺组织中生长抑制和DNA损伤修复基因45β( Gadd45β) mRNA表达情况并探讨其在沃辛瘤的形成及发展中的意义。方法搜集30例腮腺沃辛瘤组织及瘤旁正常腮腺组织。提取总RNA,然后进行逆转录... 目的:从分子学水平检测腮腺沃辛瘤相对于瘤旁腮腺组织中生长抑制和DNA损伤修复基因45β( Gadd45β) mRNA表达情况并探讨其在沃辛瘤的形成及发展中的意义。方法搜集30例腮腺沃辛瘤组织及瘤旁正常腮腺组织。提取总RNA,然后进行逆转录得到cDNA,再进行实时荧光定量PCR检测,测出CT值,通过2-△△CT的相对定量的方法对荧光定量PCR检测的CT值进行标准化,从而比较肿瘤组织及瘤旁正常腮腺组织中Gadd45βmRNA表达情况。结果 Gadd45β在正常腮腺组织及肿瘤组织中均有表达,分别为1.516±0.104和1.177±0.089, Gadd45β在沃辛瘤中表达明显下调(P<0.05)。结论 Gadd45β表达下调可作为沃辛瘤发病机理一部分。 展开更多
关键词 GADD45Β 沃辛瘤 基因表达 GADD45Β
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3种核酸提取方法及3种实时荧光定量PCR仪检测结果的比较 被引量:1
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作者 吴秋华 张勇建 +5 位作者 田真 李红东 李政 司拨云 许文波 许松涛 《中华实验和临床病毒学杂志》 CAS CSCD 2017年第2期165-168,共4页
目的 探讨不同核酸提取方法以及不同实时荧光定量PCR仪检测结果之间的差异.方法 随机挑选呼吸道、肠道病毒阳性标本各25份,采用3种方法(方法A、B、C)进行核酸提取,比较不同核酸提取方法之间的差异;随机挑选呼吸道、肠道病毒阳性核酸... 目的 探讨不同核酸提取方法以及不同实时荧光定量PCR仪检测结果之间的差异.方法 随机挑选呼吸道、肠道病毒阳性标本各25份,采用3种方法(方法A、B、C)进行核酸提取,比较不同核酸提取方法之间的差异;随机挑选呼吸道、肠道病毒阳性核酸各25份,在3种实时荧光定量PCR仪(仪器A、B、C)上进行实时荧光定量PCR,比较不同实时荧光定量PCR仪之间的差异.检测结果采用定量资料的随机区组方法分析.结果 3种核酸提取方法检测结果存在差异(x2=42.9162,P〈0.001),其中方法A、B,方法B、C之间的差异具有统计学意义(Z=7.025,P〈0.001;Z=7.9,P〈0.001),方法A、C之间的差异无统计学意义(Z=0.837,P=0.3816〉0.05).3种实时荧光定量PCR仪的检测结果也存在差异(x2=23.773,P〈0.001),其中仪器A、B,仪器A、C之间的差异有统计学意义(Z=5.70,P〈0.001;Z=6.45,P〈0.001),仪器B、C之间的差异无统计学意义(Z=0.75,P=0.4533〉0.05).结论 在相同条件下,用不同的核酸提取方法,以及用不同的实时荧光定量PCR仪检测的结果均有差异,在实际工作中要综合考虑评价检测结果. 展开更多
关键词 核酸提取 随机区组设计
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From Phenotypes to Molecules: Revolutionizing Gut Microbiota Identification Methods
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作者 WANG Xuan LV Chang-Long ZHAI Jing-Bo 《中国生物化学与分子生物学报》 CAS CSCD 北大核心 2024年第8期1065-1077,共13页
The gut microbiota is a complex ecosystem composed of many bacteria and their metabolites.It plays an irreplaceable role in human digestion,nutrient absorption,energy supply,fat metabolism,immune regulation,and many o... The gut microbiota is a complex ecosystem composed of many bacteria and their metabolites.It plays an irreplaceable role in human digestion,nutrient absorption,energy supply,fat metabolism,immune regulation,and many other aspects.Exploring the structure and function of the gut microbiota,as well as their key genes and metabolites,will enable the early diagnosis and auxiliary diagnosis of diseases,new treatment methods,better effects of drug treatments,and better guidance in the use of antibiotics.The identification of gut microbiota plays an important role in clinical diagnosis and treatment,as well as in drug research and development.Therefore,it is necessary to conduct a comprehensive review of this rapidly evolving topic.Traditional identification methods cannot comprehensively capture the diversity of gut microbiota.Currently,with the rapid development of molecular biology,the classification and identification methods for gut microbiota have evolved from the initial phenotypic and chemical identification to identification at the molecular level.This review integrates the main methods of gut microbiota identification and evaluates their application.We pay special attention to the research progress on molecular biological methods and focus on the application of high-throughput sequencing technology in the identification of gut microbiota.This revolutionary method for intestinal flora identification heralds a new chapter in our understanding of the microbial world. 展开更多
关键词 gut microbiota 16S rRNA real-time fluorescent qpcr high-throughput sequencing mass spectrum
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Application of Real-time Fluorescent Quantitative PCR in Plant
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作者 崔颖 贾晋 +2 位作者 莎娜 李俊芳 王国泽 《Agricultural Science & Technology》 CAS 2016年第2期273-278,共6页
Real-time fluorescent quantitative PCR (RQ-PCR) is a detection method by adding fluorescent dye or fluorescent probe into the PCR reaction system, using fluorescent signal accumulation to monitor amplification react... Real-time fluorescent quantitative PCR (RQ-PCR) is a detection method by adding fluorescent dye or fluorescent probe into the PCR reaction system, using fluorescent signal accumulation to monitor amplification reactions of PCR reaction process, and finally the unknown template can be quantitatively analyzed through the standard curve. So the detection level of PCR has improved from the qualitative to the quantitative. In order to provide a theoretical reference for further application, the principle, classification, advantages and disadvantages of RQ-PCR were intro- duced, and its application and progress in plants in recent years were reviewed. 展开更多
关键词 Real-time fluorescent quantitative pcr (RQ-pcr PRINCIPLE Reference gene Stress resistance of plant Transgenic product
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鸡IL-2 mRNA SYBR GreenⅠ实时荧光定量RT-PCR检测方法的建立与应用
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作者 王衡 刘博奇 宁章勇 《中国兽医学报》 CAS CSCD 北大核心 2013年第7期1073-1077,共5页
根据GenBank提供的ChIL-2参考序列设计了特异性引物,并以鸡3-磷酸甘油脱氢酶(GAPDH)基因为内参,以二者标准质粒为模板,应用SYBR GreenⅠ实时荧光定量PCR技术,并采用双标准曲线相对定量方法建立了ChIL-2mRNA荧光定量RT-PCR检测方法。结... 根据GenBank提供的ChIL-2参考序列设计了特异性引物,并以鸡3-磷酸甘油脱氢酶(GAPDH)基因为内参,以二者标准质粒为模板,应用SYBR GreenⅠ实时荧光定量PCR技术,并采用双标准曲线相对定量方法建立了ChIL-2mRNA荧光定量RT-PCR检测方法。结果显示,该方法该可以在3h左右对低拷贝量的模板(200copy/μL)进行检测,同时该检测方法具有很好的特异性和重复性。雏鸡免疫试验IL-2mRNA检测结果显示,该方法可有效应用于鸡体内IL-2在核酸水平的检测,并为今后IL-2相对定量的检测研究提供参考依据。 展开更多
关键词 鸡白细胞介素2 荧光定量RT—pcr 检测方法 双标准曲线
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血清SOD、TNF-α及荧光实时定量PCR检测在婴幼儿支原体肺炎中的应用
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作者 翟燕红 《中国妇幼保健》 CAS 北大核心 2013年第35期5830-5833,共4页
目的:探讨血清中超氧化物歧酶(SOD)、肿瘤坏死因子(TNF-α)及荧光实时定量PCR(FQ—PCR)检测在婴幼儿支原体肺炎(MPP)早期诊断和治疗中的价值。方法:选择2010年1月-2011年12月肺炎患儿136例,x光检查肺部有明显改变、血清MP抗... 目的:探讨血清中超氧化物歧酶(SOD)、肿瘤坏死因子(TNF-α)及荧光实时定量PCR(FQ—PCR)检测在婴幼儿支原体肺炎(MPP)早期诊断和治疗中的价值。方法:选择2010年1月-2011年12月肺炎患儿136例,x光检查肺部有明显改变、血清MP抗体4倍及以上滴度升高为MPP患儿,余下为非MPP患儿。分别检测患儿治疗前后的血清SOD、TNF—α浓度及对部分患儿取肺泡灌洗液后进行FQ—PCR;以80份健康儿童血清作为阴性对照。组间率的比较采用χ2检验,计量资料采用t检验,双变量相关分析采用Pearson检验。结果:治疗前MPP患儿血清中SOD和TNF—α浓度均显著高于对照组(P〈0.01)。相关分析显示,MP患儿组的血清SOD、TNF-α水平与PCR检测结果呈正相关,对照组的血清水平与PCR检测结果无相关性;治疗后MPP患儿组的SOD和TNF-α浓度水平均显著下降(P〈0.01)。结论:联合测定血清中SOD、TNF—α水平及FQ—PCR对MPP患儿的早期诊断、治疗和预后有十分重要的临床价值,可作为评价MPP的转归以及判断临床疗效的指标。 展开更多
关键词 超氧化物歧化酶 肿瘤坏死因子 荧光定量pcr 支原体肺炎
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Development of a real-time PCR assay(SYBR Green I) for rapid identification and quantification of scyphomedusae Aurelia sp.1 planulae 被引量:2
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作者 王建艳 甄毓 +2 位作者 米铁柱 于志刚 王国善 《Chinese Journal of Oceanology and Limnology》 SCIE CAS CSCD 2015年第4期974-987,共14页
The complicated life cycle ofAurelia spp., comprising benthic asexually-reproducing polyps and sexually-reproducing medusae, makes it hard for researchers to identify and track them, especially for early stage individ... The complicated life cycle ofAurelia spp., comprising benthic asexually-reproducing polyps and sexually-reproducing medusae, makes it hard for researchers to identify and track them, especially for early stage individuals, such as planulae. To solve this problem, we developed a real-time PCR assay (SYBR Green I) to identify planulae in both cultured and natural seawater samples. Species-specific primers targeting Aurelia sp.1 mitochondrial 16S rDNA (mr 16S rDNA) regions were designed. Using a calibration curve constructed with plasmids containing the Aurelia sp. 1 mt 16S rDNA fragment and a standard curve for planulae, the absolute number of mt 16S rDNA copies per planula was determined and from that the total number ofplanulae per sample was estimated. For the field samples, a 100-fold dilution of the sample DNA combined with a final concentration of 0.2 μg/μL BSA in the PCR reaction mixture was used to remove real- time PCR inhibitors. Samples collected in Jiaozhou Bay from July to September 2012 were subsequently analyzed using this assay. Peak Aurelia sp.1 planula abundance occurred in July 2012 at stations near Hongdao Island and Qingdao offshore; abundances were very low in August and September. The real-time PCR assay (SYBR Green I) developed here negates the need for traditional microscopic identification, which is laborious and time-consuming, and can detect and quantify jellyfish planulae in field plankton samples rapidly and specifically. 展开更多
关键词 Aurelia sp. 1 16S rDNA planulae real-time pcr jellyfish blooms
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A real-time PCR targeted to the upstream regions of HlyB for specific detection of Edwardsiella tarda 被引量:2
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作者 谢国驷 黄倢 +4 位作者 张庆利 韩娜娜 史成银 王秀华 刘庆慧 《Chinese Journal of Oceanology and Limnology》 SCIE CAS CSCD 2012年第5期731-737,共7页
Edwardsiella tarda has become one of the most important emerging pathogens in aquaculture industry. Therefore, a rapid, reproducible, and sensitive method for detection and quantification of this pathogen is needed ur... Edwardsiella tarda has become one of the most important emerging pathogens in aquaculture industry. Therefore, a rapid, reproducible, and sensitive method for detection and quantification of this pathogen is needed urgently. To achieve this purpose, we developed a TaqMan-based real-time PCR assay for detection and quantification orE. tarda. The assay targets the hemolysin activator HlyB domain protein of E. tarda. Our optimized TaqMan assay is capable of detecting as little as 40 fg of genomic DNA per reaction. A standard curve was generated from the threshold cycle values (y) against log10 (E. tarda genomic DNA concentration) as x. The intra- and inter-assay coefficient of variation (CV) values were less than 2.06% and 1.05% respectively, indicating that the assay had good reproducibility. This method is highly specific to E. tarda strains, as it shows no cross-reactivity to Edwardsiella ictaluri, a member of the same genus, or to nine other fish-pathogenic bacteria species belonging to three other genera. This sensitive and specific real-time PCR assay provides a valuable tool for diagnostic quantitation of E. tarda in clinical samples. 展开更多
关键词 Edwardsiella tarda TAQMAN real-time pcr DETECTION
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Detection of Syndecan-1 and Heparanase-1 Genes in Esophageal Carcinoma by Quantitative RT-PCR 被引量:1
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作者 Jun-li SI Yu-qin QI +3 位作者 Jing-yuan CUI Song-mei WANG He WANG Mei LU 《Clinical oncology and cancer researeh》 CAS CSCD 2010年第4期253-258,共6页
OBJECTIVE To quantitatively explore the expression of Syndecan-1 and heparanase-1 in esophageal cancer tissue as well as their relationship with the clinicopathological factors, in order to evaluate their roles in tum... OBJECTIVE To quantitatively explore the expression of Syndecan-1 and heparanase-1 in esophageal cancer tissue as well as their relationship with the clinicopathological factors, in order to evaluate their roles in tumor invasion and metastasis.METHODS Real-time fluorescence quantitative PCR (Q-PCR) was used to analyze the expression levels of Syndecan-1 and heparanase-1 genes£?participants included 67 cases with esophageal cancers and 32 healthy volunteers.RESULTS The expression of Heparanase-1 gene in esophageal cancers was higher than that in normal esophageal tissue (P 〈 0.001), and the expression of Syndecan-1 gene in the normal esophageal tissue was higher compared with esophageal cancers (P 〈 0.001). The positive rates of Syndecan-1 and Heparanase-1 gene in esophageal cancer were 13.4% (9/67) and 85.1% (57/67).The expression of Syndecan-1 and Heparanase-1 genes was signifi cantly related to di. erentiation, depth of infi ltration, lymph node metastasis, vessel metastasis, and TNM stages of disease (P 〈 0.05). In an attempt to measure the association between the 2 agents, this study found that the expression of Syndecan-1 mRNA had a significantly negative correlation with the expression of Heparanase-1 mRNA by using Spearman rank correlation test (OR = -0.572, P 〈 0.001).CONCLUSION Syndecan-1 and Heparanase-1 play important roles in the invasion and metastasis of esophageal cancer. The reduction of Syndecan-1 and/or the increase of Heparanase-1 may influence the invasion and metastasis of malignant tumors.Thus the combination assay of Syndecan-1 and Heparanase-1 may contribute to the diagnosis and treatment of malignant tumors. 展开更多
关键词 SYNDECAN-1 esophageal neoplasms neoplasm invasiveness neoplasm metastasis pcr
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Detection of Survivin mRNA in nasopharyngeal carcinoma by real-time fluorescence quantitative RT-PCR 被引量:1
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作者 Shengmiao Fu Junhong Cai +5 位作者 Zhihua Tu Yutian Wang Liqun Deng Zhu Liang Zhenqun Lin Xuanju Gong 《The Chinese-German Journal of Clinical Oncology》 CAS 2008年第9期523-526,共4页
Objective: To establish the method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in nasopharyngeat carcinoma (NPC) tissues. Methods: The total RNA was extracted from N... Objective: To establish the method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in nasopharyngeat carcinoma (NPC) tissues. Methods: The total RNA was extracted from NPC cell line CNE-2 and tissues with Trizol and then been transcribed reversely to cDNA, a method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in NPC tissues had been established, in which chronic nasopharyn-gitis patients' nasopharynx tissues treated as control group. Results: The expression of Survivin mRNA all could be detected either in CNE-2 cells, NPC tissues or in chronic nasopharyngitis patients' nasopharynx tissues, and there was higher the expression level of Survivin mRNA in NPC tissues than which in chronic nasopharyngitis patients' nasopharynx tissues, the difference was significant (P 〈 0.01). The expression of Survivin mRNA could be detected both in stage Ⅰ + Ⅱ and stage Ⅲ + Ⅳ NPC, and there was no significant difference in relative quantifications of gene expression between these two groups (P 〉 0.05). There was no relationship between Survivin mRNA expression and age and sex of NPC patients (P 〉 0.05). Conclusion: Real time fluorescence quantitative RT-PCR is a rapid, effective and high sensitive method for detecting the expression of Survivin mRNA in NPC tissues. The overexpression of Survivin mRNA may play some roles in pathogenesis of NPC. 展开更多
关键词 nasopharyngeal carcinoma (NPC) real-time fluorescence quantitative RT-pcr gene expression apoptosisinhibitor Survivin
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Detection of PRL-2 gene expression in hepatocellular carcinoma by real-time fluorescence quantitative PCR
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作者 Chao Cheng Amos Ela Bella +2 位作者 Ailin Guo Guoyong Wu Weikang Wu 《The Chinese-German Journal of Clinical Oncology》 CAS 2009年第4期210-213,共4页
Objective:To quantitatively detect the expression level of PRL-2 in primary hepatocellular carcinoma using real-time fluorescence quantitative PCR.Methods:Total RNA isolated from human HCC and liver tissue adjacent to... Objective:To quantitatively detect the expression level of PRL-2 in primary hepatocellular carcinoma using real-time fluorescence quantitative PCR.Methods:Total RNA isolated from human HCC and liver tissue adjacent to the tumor was reversely transcribed into cDNA.Real-time fluorescence quantitative PCR(Q-PCR) method was used to analyze the expres-sion level of PRL-2 gene.Results:The Q-PCR method was performed successfully to precisely detect RNA level.PRL-2 was expressed in all portal vein tumor thrombosis(PVTT) and HCC,but only in some paratumor tissue.The highest expression level of PRL-2 gene was recorded in PVTT;meanwhile expression level of PRL-2 was higher than that in paratumor liver tis-sues and in HCC(P < 0.01),and it was higher in HCC than that in paratumor liver tissues.Conclusion:The Q-PCR may be the most precise method to quantitatively detect RNA level and can be used in gene expression changes.The PRL-2 gene has higher expression in PVTT than that in HCC and in paratumor liver tissue cells,indicating that it plays an important role in the development and metastasis of the HCC. 展开更多
关键词 CARCINOMA HEPATOCELLULAR polymerase chain reaction PRL2 gene expression
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Selection of Reference Genes in Saccharopolyspora Spinosa for Real-Time PCR
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作者 张传波 薛超友 +1 位作者 申月琪 卢文玉 《Transactions of Tianjin University》 EI CAS 2015年第5期461-467,共7页
Reverse transcription quantitative PCR (RT-qPCR) combined with the published genome information of Saccharopolyspora spinosa can allow sophisticated studies about S. spinosa, including Studying the regulation of spi... Reverse transcription quantitative PCR (RT-qPCR) combined with the published genome information of Saccharopolyspora spinosa can allow sophisticated studies about S. spinosa, including Studying the regulation of spinosyn biosynthesis, finding new target genes for engineering, and discovering and exploiting other macrolide secondary metabolites. Studies have demonstrated that appropriate internal control is needed to normalize target genes at transcription levels. However, many studies have shown that no single reference gene is universal for all strains under all experimental conditions. Thus, eight candidate reference genes of three different S. spinosa strains in two different cultures were studied to find suitable reference gene(sl. The number of amplification cycles of these candidate genes was calculated by BestKeeper, NormFinder and geNorm. The results indicated that the most suitable reference genes for normalization during the fermentation of S. spinosa were 16S rRNA and rbL13. 展开更多
关键词 real-time pcr reference genes SaecharopOlysporaspinosa
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SOCS-3在大鼠慢性肝损伤中的表达研究 被引量:5
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作者 张千 邓存良 《四川医学》 CAS 2014年第9期1126-1128,共3页
目的通过研究细胞因子信号转导抑制因子-3(Suppressor of cytokine signaling-3,SOCS-3)在慢性肝损伤中的表达,为阐明其在慢性肝损伤中的作用提供实验基础。方法采用CCL4法构建大鼠慢性肝损伤模型,通过实时荧光定量PCR、免疫组化检测大... 目的通过研究细胞因子信号转导抑制因子-3(Suppressor of cytokine signaling-3,SOCS-3)在慢性肝损伤中的表达,为阐明其在慢性肝损伤中的作用提供实验基础。方法采用CCL4法构建大鼠慢性肝损伤模型,通过实时荧光定量PCR、免疫组化检测大鼠肝脏组织SOCS-3的表达。结果慢性肝损伤组SOCS-3 mRNA相对表达量及SOCS-3蛋白高于对照组,差异有统计学意义(P<0.05)。结论在慢性肝损伤中,随着肝脏损伤加重,因炎症因子持续刺激,SOCS-3表达逐渐增加,提示SOCS-3在慢性肝损伤发生发展过程中起重要作用。 展开更多
关键词 细胞因子信号转导抑制因子-3 慢性肝损伤 时荧光定量pcr
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宗地花猪心脏型脂肪酸结合蛋白基因的表达 被引量:1
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作者 李平 燕志宏 +1 位作者 张芸 陈伟 《贵州农业科学》 CAS 2015年第4期146-149,共4页
为有效提高宗地花猪的肉品质,合理评价养殖模式提供理论依据,参照GenBank中猪H-FABP基因序列设计引物,以猪18SRNA为内参基因,应用实时荧光定量PCR方法检测H-FABP基因在不同饲养条件下宗地花猪不同组织器官中的表达量。结果表明:H-FABP... 为有效提高宗地花猪的肉品质,合理评价养殖模式提供理论依据,参照GenBank中猪H-FABP基因序列设计引物,以猪18SRNA为内参基因,应用实时荧光定量PCR方法检测H-FABP基因在不同饲养条件下宗地花猪不同组织器官中的表达量。结果表明:H-FABP基因在肺脏、心脏、肾脏、小肠、脾脏、肝脏、背最长肌、腰大肌中均有表达,但不同饲养条件下其表达水平各异,放养型背最长肌>腰大肌>肾脏>心脏>小肠>肺脏>脾脏>肝脏,背最长肌中表达量显著高于其他组织(P<0.05);圈养型腰大肌>脾脏>心脏>肺脏>小肠>肝脏>背最长肌>肾脏,腰大肌中表达量显著高于其他组织(P<0.05),肝脏中表达量高于背最长肌(P>0.05)。两种饲养条件下宗地花猪肺脏、肝脏中表达量相当,而在心脏、肾脏、小肠、脾脏、背最长肌中表达差异较大。 展开更多
关键词 H-FABP基因 宗地花猪 时荧光定量pcr
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嗜热四膜虫两类不同金属硫蛋白的功能补偿分析 被引量:5
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作者 卢剑功 许静 +1 位作者 张鹏幸 王伟 《水生生物学报》 CAS CSCD 北大核心 2014年第2期249-256,共8页
为了分析嗜热四膜虫两类金属硫蛋白之间的关系,研究分别构建了MTT1-MTT3和MTT2-MTT4的基因敲除载体,通过同源重组获得敲除大核MTT1-MTT3和MTT2-MTT4的两种嗜热四膜虫突变体细胞株△MTT1-MTT3和△MTT2-MTT4。两种突变体细胞株暴露在Cd2+... 为了分析嗜热四膜虫两类金属硫蛋白之间的关系,研究分别构建了MTT1-MTT3和MTT2-MTT4的基因敲除载体,通过同源重组获得敲除大核MTT1-MTT3和MTT2-MTT4的两种嗜热四膜虫突变体细胞株△MTT1-MTT3和△MTT2-MTT4。两种突变体细胞株暴露在Cd2+、Cu2+和H2O2的生长表现出显著不同,△MTT1-MTT3突变体细胞对Cd2+的耐受性显著下降,而△MTT2-MTT4突变体细胞对Cu2+和H2O2的耐受性均显著下降。实时荧光定量PCR分析不同突变体中其他MTT基因的表达变化,在△MTT2-MTT4突变体细胞株中,MTT5的表达水平下调,在500μmol/L Cu2+处理后,△MTT2-MTT4突变体细胞中MTT1、MTT3和MTT5表达相对野生型分别上调6.1、9.5和8.5倍。在△MTT1-MTT3突变体细胞中,MTT2、MTT4和MTT5的表达水平下调,当5μmol/L Cd2+处理后,△MTT1-MTT3突变体细胞株MTT5表达水平相对野生型上调2.9倍,而MTT2和MTT4表达水平相对野生型分别下降了4.9倍和2.5倍。结果表明嗜热四膜虫中的金属硫蛋白MTT1、MTT3和MTT5主要参与细胞的重金属解毒功能;而MTT2和MTT4主要参与细胞内正常的新陈代谢功能,不同的金属硫蛋白基因之间的表达存在相互调控和功能补偿。 展开更多
关键词 嗜热四膜虫 金属硫蛋白 基因敲除
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