【作者】 董晓丽；

【导师】 王加启； 赵国琦；

【作者基本信息】 扬州大学 ， 动物营养与饲料科学， 2009， 硕士

【摘要】 本试验由三部分组成,主要研究了不同泌乳期小鼠乳腺组织、经过不同免疫处理的小鼠肝脏组织以及幼龄小鼠成长过程中小肠组织内参基因的稳定性。应用荧光实时定量PCR检测基因B2M, ACTB, GAPDH, SDHA, HPRT1和ARBP的表达水平,并应用geNorm程序进行分析,最终选出合适的内参基因,为研究目标基因的表达奠定基础。试验一:实时定量PCR(qPCR)是检测细胞和组织中mRNA表达量最常用的技术之一,而使用qPCR要求对数据进行标准化。在本试验中通过qPCR研究六个潜在内参基因(B2M, ACTB, GAPDH, SDHA, HPRT1和ARBP)在不同泌乳期小鼠乳腺组织中的表达情况。经过SAS6.12中ANOVA模型进行统计分析,结果表明B2M差异显著(P<0.05)。经过geNorm程序分析,所选内参基因稳定性从高到低排序分别是GAPDH/HPRT1, ARBP, ACTB, SDHA, B2M。由此推荐应用基因GAPDH和HPRT1作为实时定量PCR不同泌乳期小鼠乳腺组织的内参照。试验二:目前实时定量PCR技术已广泛应用于细胞或组织mRNA的转录水平的检测和定量。选择合适的内参基因可以消除不同标本在RNA的产量、质量以及逆转录效率上可能存在的差别,从而获得目标基因特异性表达的真正差异。本试验应用实时定量PCR技术,研究小鼠在经过免疫刺激后,B2M, ACTB, GAPDH, SDHA, HPRT1和ARBP共6个内参基因在肝脏组织中的表达情况。结果表明,这6个内参基因表达存在差异。经过geNorm程序统计学分析,确定了ACTB, GAPDH两个看家基因适用于校正目标基因的表达量,为研究小鼠免疫刺激后肝脏目标基因的表达奠定基础。试验三:内参基因常用于实时定量PCR检测mRNA表达水平的校正和标准化,但是内参基因表达受生理阶段、组织或细胞以及实验条件的影响。因此,本试验选择B2M, ACTB, GAPDH, SDHA, HPRT1和ARBP共6个内参基因,研究其在幼龄小鼠小肠组织内的表达情况。经过geNorm程序和NormFinder程序分析,最终确定了内参基因SDHA和HPRT1适合用于校正目标基因的表达量,为研究幼龄小鼠小肠组织基因表达奠定基础。更多还原

【Abstract】 This research composed of three experiments which were carried out to study the stability of reference genes in mammary gland of mouse in different lactation period, in liver of mouse after immunity treatment and in small intestine of growing mouse. The expression levels of B2M, ACTB, GAPDH, SDHA, HPRT1 and ARBP genes were detected and quantified by real-time PCR respectively. Data were analysed subsequently by geNorm algorithm, and then the suitable reference genes were chosen and applied usefully for the research of target gene in mouse.EXPERIMENT 1:Real-time quantitative PCR (qPCR) is one of the most widely used techniques for detection and quantification of mRNA expression in cells or tissues. The use of qPCR requires data normalization using internal standards such as housekeeping genes (HKGs) to obtain accurate results because of potential analytical errors due to variation. In this study, the expression levels of six potential reference genes (B2M, ACTB, GAPDH, SDHA, HPRT1 and ARBP) were investigated in mouse mammary gland by real-time qPCR using SYBR green during the different lactation days. Data were analyzed by ANOVA procedure of SAS (Version 6.12, SAS). The results showed that the expression of B2M exhibited a significant difference (P<0.05). The ranking of expression stability in these genes was (from the most stable to the least stable): GAPDH/HPRT1, ARBP, ACTB, SDHA, B2M by means of geNorm algorithm. This study suggested that the two genes GAPDH and HPRT1 may be recommented as references for normalization of real-time qPCR in mammary gland of mice in different lactation days. EXPERIMENT 2:Real-time quantitative PCR (RT-PCR) is the techniques of choice for detection and quantification of mRNA expression in cells or tissues. Suitable reference gene is essential for high precision of target gene by taking the RNA quality and efficiencies of reverse transcription into account. The purpose of the present study was to investigate the expression of B2M, ACTB, GAPDH, SDHA, HPRT1 and ARBP in mouse liver after immunity treatment by RT-PCR individually. Differences in expression levels were observed by geNorm analysis. ACTB and GAPDH were determined as suitable internal control genes. The resuts implied ACTB and GAPDH could be applied to compare the expression of target gene in mouse liver after immunity treatment.EXPERIMENT 3: Reference genes are widely used for normalization of the expression levels of mRNA in RT-PCR. But the expression of reference gene is influenced by physiological stage, tissues or cells, and the condition of experiment. The aims of this experiment were to investigate the expression of B2M, ACTB, GAPDH, SDHA, HPRT1 and ARBP by RT-PCR in small intestine of young mouse. By geNorm and normFinder analysis, SDHA and HPRT1 were finally determined as suitable reference genes used to normalize mRNA levels between different samples. The two chosen genes could be useful for research of target gene in small intestine of growing mouse.更多还原