共查询到19条相似文献,搜索用时 187 毫秒
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实时荧光定量PCR (FQ-PCR)标准曲线法准确定量基因表达的关键在于标准品与待检样本的扩增效率是否一致. 为检测DNA标准品与样本cDNA扩增效率的一致性,探讨定量用标准品的最佳制备方法,本研究以脂肪酸结合蛋白5(Fabp5)、过氧化物酶体增殖活化受体α (Ppar-α)及β肌动蛋白(β-Actin)的3个基因为对象,分别采用质粒纯化法、PCR产物直接纯化法、PCR产物凝胶回收法制备DNA标准品,10倍梯度稀释后用FQ PCR制作标准曲线. 并以10倍梯度稀释的样本cDNA标准曲线的参数为对照,进行比较分析. 结果表明,不同方法制备的DNA标准品的扩增效率差异较大,并且与cDNA的扩增效率不一致,不能对cDNA样本进行准确定量. 另外,虽然目的基因在cDNA样本中的拷贝未知,不能对基因表达水平进行绝对定量,但因不同cDNA样本的同一基因的扩增效率一致, 可对基因的表达进行准确的相对定量. 相似文献
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SYBR Green Ⅰ实时荧光定量RT-PCR测定猴免疫缺陷病毒RNA拷贝数方法的建立 总被引:6,自引:0,他引:6
目的 建立SYBR Green Ⅰ荧光染料实时定量RT-PCR方法,测定猴免疫缺陷病毒(SIV)RNA拷贝数.方法 巢式RT-PCR扩增SIV病毒RNA gag基因上1360-1837之间的长度为477 bp的片段,将该片段克隆到pGEM T载体上,构建pGEM-SIVgag477质粒.该质粒经限制性内切酶Not I酶切后,进行体外转录,转录出的RNA产物(RS)纯化后10倍系列稀释,作出标准曲线,作为SIV病毒RNA荧光定量检测的外标准品.结果 应用Qiagen公司QuantiTect SYBR GREEN RT-PCR Kit,该标准品可精确定量到100 copies/μL.结论 制备的RS外标准品纯度高,SYBR Green Ⅰ荧光染料实时定量RT-PCR法特异性、敏感性高,稳定性好,可用于定量测定猴免疫缺陷病毒(SIV)RNA拷贝数. 相似文献
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SYBR Green I实时荧光定量RT-PCR测定猴免疫缺陷病毒RNA拷贝数方法的建立 总被引:2,自引:0,他引:2
目的建立SYBR Green I荧光染料实时定量RT-PCR方法,测定猴免疫缺陷病毒(SIV)RNA拷贝数。方法巢式RT-PCR扩增SIV病毒RNAgag基因上1360-1837之间的长度为477 bp的片段,将该片段克隆到pGEMT载体上,构建pGEM-SIVgag477质粒。该质粒经限制性内切酶NotⅠ酶切后,进行体外转录,转录出的RNA产物(RS)纯化后10倍系列稀释,作出标准曲线,作为SIV病毒RNA荧光定量检测的外标准品。结果应用Qiagen公司QuantiTect SYBR GREEN RT-PCR Kit,该标准品可精确定量到100 copies/μL。结论制备的RS外标准品纯度高,SYBR Green I荧光染料实时定量RT-PCR法特异性、敏感性高,稳定性好,可用于定量测定猴免疫缺陷病毒(SIV)RNA拷贝数。 相似文献
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以无芒隐子草干旱诱导的cDNA文库中获得的SAMDC序列为基础,应用实时荧光定量RT-PCR技术同时结合相对定量和绝对定量方法,以分析无芒隐子草SAMDC基因在不同组织、不同胁迫梯度下的表达为案例,详细介绍并比较相对定量和绝对定量在基因表达方面的应用.分别采集无芒隐子草幼苗自然干旱0,2,4,6,8,10d以及恢复浇水1,4d的叶片和根系材料,相对定量采用2-ΔΔCT.法;以相应基因的质粒扩增产物为标准品制作标准曲线,对定量PCR的引物浓度进行了优化,而后筛选最稳定的内参基因,以内参基因获得的标准化因子对绝对定量数据进行标准化.相对定量2-ΔΔCT法结果显示CsSAMDC基因在干旱胁迫8d后,叶片中的表达量为胁迫前的0.62±0.13倍,根中的表达量为胁迫前的5.93±0.71倍.绝对定量表明,CsSAMDC基因只在胁迫第8d和第10d的根组织中检测到表达量显著增大,胁迫第4~6d和恢复浇水后1~4d,CsSAMDC基因在叶和根组织中的表达较对照降低.相对定量和绝对定量在DsSAMDC基因表达研究中显示相对一致的趋势. 相似文献
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粪便中肠球菌SYBR GreenI荧光定量PCR检测方法的建立 总被引:2,自引:0,他引:2
目的利用SYBR GreenI荧光定量PCR方法,建立肠球菌实时荧光PCR检测方法,并初步应用于粪便中肠球菌的检测。方法根据GenBank发表的肠球菌23S rRNA基因序列的保守区域设计合成特异性的引物;利用构建的质粒标准品绘制两种标准曲线,构建基因拷贝数、细菌数为分析指标的定量分析模型并初步应用于粪便标本的检测分析。结果所建立的SYBR GreenI荧光定量PCR方法检测灵敏度可达7个拷贝数/reaction。粪便样本根据实时荧光定量PCR方法所得的理论数值与培养菌值之间差异无显著性(P>0.05)。非炎性腹泻标本中菌数与健康成人标本中菌数差异无显著性(P>0.05)。灵敏度曲线所得的数值大于菌数标准曲线,可能由于DNA提取过程中存在部分的损失。检测粪便标本结果显示SYBR GreenI荧光定量PCR方法较平板计数法敏感、快捷、简便。结论本研究建立了一种灵敏、特异、简便易行的肠球菌定量检测方法。 相似文献
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目的:建立茶树α-tubulin基因实时荧光定量RT-PCR方法。方法:根据GenBank中茶树α-tubulin基因保守区域设计一对特异性引物,将PCR扩增得到的α-tubulin基因克隆到pTG19-T载体上,构建的重组质粒标准品经1/10梯度稀释后,用SYBR GreenⅠ染料法绘制标准曲线,并进行融解曲线分析。结果:标准曲线Ct值检测范围为14.56~27.09,相关系数为0.991,溶解曲线分析显示产物为特异的单峰,其Tm值为81±0.3℃。结论:建立了茶树α-tu bulin基因实时荧光定量RT-PCR法,为以α-tubulin作为茶树内参基因进行功能基因表达差异研究奠定了基础。 相似文献
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荧光定量PCR数据处理方法的探讨 总被引:23,自引:0,他引:23
real time RT-PCR通过直接检测指数扩增期的PCR产物来确定基因的初始模板量,是定量基因表达最灵敏的方法,其准确性取决于扩增效率以及数据的处理方法。目前最常用的两种分析荧光定量PCR实验数据的方法是绝对定量和相对定量。绝对定量方法是通过标准曲线计算起始模板的拷贝数;相对定量方法则是比较处理过的样品和未处理过的样品目的基因之间的表达差异。而相对定量又有双标准曲线法,2^-△△α法以及动力学法。该文介绍了这些方法的推导、假设及其应用。 相似文献
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目的 采用实时荧光定量PCR检测白天和晚上所形成牙菌斑生物膜中变异链球菌的数量,比较早晚牙菌斑中变异链球菌定植的差异.方法 收集30名健康成人全口口腔洁治后白天和晚上形成的龈上菌斑,提取细菌基因组DNA.合成变异链球菌特异性引物,纯化PCR产物获得目的基因连接于pTA-TA载体,克隆于大肠埃希菌DH5 α感受态细胞.选取阳性克隆测序后纯化质粒DNA,获得质粒标准品.将样本和梯度稀释的质粒标准品进行SYBR Green Ⅰ实时荧光定量PCR检测,确定标准曲线,定量样本中变异链球菌DNA的拷贝数.结果 早晚牙菌斑细菌基因组DNA样本的扩增曲线均在标准品的扩增曲线范围内.晚上所形成牙菌斑中定植的变异链球菌数量(对数值7.67 ±0.77)高于白天定植的变异链球菌数量(对数值7.25±0.62)(P =0.007).结论 牙菌斑的微生物定植存在日夜节奏变化,晚上所形成牙菌斑中变异链球菌数量多于白天. 相似文献
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抗凝血酶Ⅲ转基因山羊外源基因拷贝数及其蛋白表达量的测定 总被引:1,自引:0,他引:1
目的:检测抗凝血酶Ⅲ(ATⅢ)转基因山羊外源基因的拷贝数及ATⅢ蛋白的表达量,分析拷贝数与蛋白表达量的相关性。方法:用Primer 5.0软件设计外源基因ATⅢ及山羊管家基因GAPDH的引物,以倍比稀释的标准品进行实时荧光定量PCR,制作标准曲线,通过标准曲线计算外源基因拷贝数;通过酶显色底物法检测ATⅢ蛋白的含量。结果:依据ATⅢ和GAPDH基因标准曲线方程计算得到的山羊个体间外源基因的拷贝数相差很大,最少的为5,最多为25,且不同转基因山羊间ATⅢ表达水平的差别达10倍以上。结论:外源基因表达水平在受到拷贝数的影响外,也受到整合位点等其他因素的重要影响。 相似文献
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目的应用NASBA方法制备SIV/SHIV RNA定量测定标准品。方法应用NASBA方法直接扩增SIVmac251病毒gag基因上1476~1685之间的片段,扩增的RNA产物(RS-NASBA)纯化后10倍系列稀释,测定定量曲线、标准曲线,测定该标准品的稳定性和重复性。结果应用Qiagen公司QuantiTect SYBR GREEN RT-PCRKit,该标准品可精确定量到2.033×10 copies/μL。结论外标准品RS.NASBA纯度高,稳定性好,可用于定量测定SIV/SHIV RNA拷贝数。 相似文献
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Genomic DNA functions as a universal external standard in quantitative real-time PCR 总被引:2,自引:0,他引:2
Yun JJ Heisler LE Hwang II Wilkins O Lau SK Hyrcza M Jayabalasingham B Jin J McLaurin J Tsao MS Der SD 《Nucleic acids research》2006,34(12):e85
Real-time quantitative PCR (qPCR) is a powerful tool for quantifying specific DNA target sequences. Although determination of relative quantity is widely accepted as a reliable means of measuring differences between samples, there are advantages to being able to determine the absolute copy numbers of a given target. One approach to absolute quantification relies on construction of an accurate standard curve using appropriate external standards of known concentration. We have validated the use of tissue genomic DNA as a universal external standard to facilitate quantification of any target sequence contained in the genome of a given species, addressing several key technical issues regarding its use. This approach was applied to validate mRNA expression of gene candidates identified from microarray data and to determine gene copies in transgenic mice. A simple method that can assist achieving absolute quantification of gene expression would broadly enhance the uses of real-time qPCR and in particular, augment the evaluation of global gene expression studies. 相似文献
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目的建立SYBR GreenⅠ荧光染料实时定量RT-PCR方法,测定实验动物等来源的EV71病毒RNA。方法运用EV71VP1保守区引物,优化real time RT-PCR条件,运用NASBA方法扩增EV71病毒RNA,计算拷贝数,经10倍系列稀释做出标准曲线,作为EV71病毒RNA定量检测的外标准品。结果应用Qiagen公司QuantiTect SYBR Green RT-PCR Kit,该标准品可精确定量到100copies/μL,PCR扩增效率达到99.5%。结论 SYBRGreenⅠ荧光染料实时定量PCR法测定EV71病毒RNA拷贝数的方法敏感性高、稳定性好,可用于EV71病毒RNA载量的定量测定。 相似文献
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Analyzing real-time PCR data by the comparative C(T) method 总被引:26,自引:0,他引:26
Two different methods of presenting quantitative gene expression exist: absolute and relative quantification. Absolute quantification calculates the copy number of the gene usually by relating the PCR signal to a standard curve. Relative gene expression presents the data of the gene of interest relative to some calibrator or internal control gene. A widely used method to present relative gene expression is the comparative C(T) method also referred to as the 2 (-DeltaDeltaC(T)) method. This protocol provides an overview of the comparative C(T) method for quantitative gene expression studies. Also presented here are various examples to present quantitative gene expression data using this method. 相似文献
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Quantitative real-time or kinetic RT-PCR is increasingly used for the quantification of specific mRNA targets, especially in clinical applications. To quantify the mRNA of cytokines and their receptors, which play important roles in the pathogenesis of autoimmune diseases such as multiple sclerosis, we have developed quantitative two-step RT-PCR assays for IL-4, IL-4R, IFN-gamma, IFN-beta, and the housekeeping gene porphobilinogen deaminase (PBGD). The LightCycler system was used to quantify the copy numbers with the sequence-specific hybridization probe detection format. The quantification was carried out on the basis of standard curves generated with external homologous plasmids for each different parameter in relation to the gene expression of PBGD. Therefore, this procedure represents a relative quantification method with external standards, as the standard curves were used to obtain an absolute value for the copy numbers of the targets and the reference (PBGD). The new software version 3.5 of the LightCycler system allows the construction of a single parameter-dependent plasmid standard curve for the quantification of unknown samples from different runs. Here we demonstrate how to achieve precise and reproducible quantification, even when using measurements from different PCR runs. 相似文献
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Amine-reactive isobaric tagging reagents such as iTRAQ (isobaric tags for relative and absolute quantitation) have recently become increasing popular for relative protein quantification, cell expression profiling, and biomarker discovery. This is due mainly to the possibility of simultaneously identifying and quantifying multiple samples. The principles of iTRAQ may also be applied to absolute protein quantification with the use of synthetic peptides as standards. The prerequisites that must be fulfilled to perform absolute quantification of proteins by iTRAQ have been investigated and are described here. Three samples of somatropin were quantified using iTRAQ and synthetic peptides as standards, corresponding to a portion of the protein sequence. The results were compared with those obtained by quantification of the same protein solutions using double exact matching isotope dilution mass spectrometry (IDMS). To obtain reliable results, the appropriate standard peptides needed to be selected carefully and enzymatic digestion needed to be optimized to ensure complete release of the peptides from the protein. The kinetics and efficiency of the iTRAQ derivatization reaction of the standard peptides and digested proteins with isobaric tagging reagents were studied using a mixture of seven synthetic peptides and their corresponding labeled peptides. The implications of incomplete derivatization are also presented. 相似文献