鳜鱼基因表达转录分析中的内参选择比较

目前基因表达的转录分析多采用单一或多个看家基因作为内参来校正目的基因的表达量。该实验以鳜鱼6个不同组织和5个不同胚胎发育阶段为研究对象,应用实时荧光定量PCR技术,观察了GAPDH、β-actin和18S rRNA三个看家基因mRNA水平的表达情况。geNorm统计分析表明,胚胎发育阶段β-actin表达最为稳定;不同的组织样品间,GAPDH表达最为稳定;而18S rRNA 的表达在不同的发育阶段不稳定。当利用多基因作为内参时,使用两个最稳定表达的看家基因即可对目的基因的表达进行准确校正。该结果证实了基因表达转录分析中内参基因选择的必要性,同时为鳜鱼等鱼类基因表达分析时内参基因的选择提供有价值的参考     

定量PCR检测microRNA表达的数据归一化新技术

数据归一化技术对研究结果的分析具有重要的作用.在定量PCR实验中,通常利用稳定表达的看家基因作为实验数据归一化的内参,但最近的研究表明,这些看家基因的表达量在不同的生理病理过程中也表现出显著的变化,不适合作为数据归一化处理的基准.针对这一问题,提出一种新的数据处理技术,利用单细胞归一化方法(percellome),对定量PCR检测miRNA表达的数据进行处理,显著提高了数据处理的准确性.以8周龄/40周龄小鼠脑为实验材料,选择14种microRNA的表达情况进行了检测.在研究中,将3种不同拷贝数的人工合成的RNA片段(spike RNA)作为内参加入到样品中,用于microRNA表达检测的归一化基准.研究发现,未经处理的microRNA单细胞拷贝数的变化范围为2.0×105～4.3×105,而经单细胞归一处理,上述表达变化范围为2到26倍.该项研究还发现,看家基因U6 ncRNA和5S rRNA的表达水平存在显著的变化,在以基因组DNA为归一化基准时,其表达量变化为1.5和4.8倍,而在以RNA水平为归一化基准时,其表达量变化为5.8和3.8倍,表明这些基因不适合作为数据处理的基准.据此,为microRNA的定量研究提供了一种新的、可靠的归一化方案.     

草鱼MYH mRNA表达量分析中采用的内参基因稳定性比较

本研究分别以β-actin、18S rRNA和GAPDH为内参基因,采用实时荧光定量PCR对草鱼早期发育时期肌球蛋白重链(myosin heavy light,MYH)基因的mRNA表达量进行分析,并比较不同内参基因对MYH基因mRNA表达水平检测结果的准确性.研究结果表明,以β-actin和GAPDH作为内参,MYH基因mRNA表达水平完全一致,其表达量从原肠到仔鱼阶段逐次递增,仔鱼与原肠期阶段相比表达量差异显著;当采用18S rRNA作为内参时,MYH基因mRNA在发育阶段的表达量呈不稳定状态.因此,β-actin和GAPDH均可作为内参基因,用于草鱼早期发育中MYH基因mRNA的相对定量研究:而18S rRNA作为内参时,可能会对检测结果造成偏差.本研究不仅准确的揭示了草鱼MYH基因mRNA的表达特征,并且为荧光定量PCR技术在鱼类基因表达研究方面提供了有价值的参考.     

Validation of internal control genes for quantitative gene expression studies in chickpea (Cicer arietinum L.)

The real-time polymerase chain reaction (PCR) data requires normalization with an internal control gene expressed at constant levels under all the experimental conditions being analyzed for accurate and reliable gene expression results. In this study, the expression of 12 candidate internal control genes, including ACT1, EF1α, GAPDH, IF4a, TUB6, UBC, UBQ5, UBQ10, 18SrRNA, 25SrRNA, GRX and HSP90, in a diverse set of 18 tissue samples representing different organs/developmental stages and stress conditions in chickpea (Cicer arietinum L.) has been validated. Their expression levels vary considerably in various tissue samples analyzed. The expression levels of EF1α and HSP90 are most constant across various organs/developmental stages analyzed. Similarly, the expression levels of IF4a and GAPDH are most constant across various stress conditions. A set of two most stable genes is found sufficient for accurate and reliable normalization of real-time PCR data in the given set of tissue samples of chickpea. The genes with most constant expression identified in this study should be useful for normalization of gene expression data in a wide variety of tissue samples in chickpea.     

Variation of Expression Levels of Seven Housekeeping Genes at Different Life-History Stages in Porphyra yezoensis

In order to identify the optimal internal control for relative real-time PCR when studying target gene expression in the red alga Porphyra yezoensis, we quantified the expression of seven housekeeping genes (18S ribosomal RNA, 30S ribosomal protein S8, Polyubiquitin-2, Glyceraldehyde-3-phosphate dehydrogenase, Elongation factor 1-alpha, Beta-tubulin and Actin 3) at different life-history stages. Absolute quantification was done by normalization to total RNA quantity and by normalization to genomic DNA quantity. We used these two normalization approaches, comparing the differences of expression levels of all candidate housekeeping genes between any two generations and across three life-history stages (filamentous sporophytes, leafy gametophytes and conchospores). We found GAPDH had the best stability in all cases and we recommend that GAPDH be considered as a potential internal control for gene expression studies at different life-history stages in P. yezoensis.     

放射诱导基因FHL2在人群中表达水平的检测

目的：分析FHL2基因在健康人群个体间表达的差异,评估FHL2作为一种新的辐射生物剂量计的可行性。方法：收集20例健康人血液样本,提取白细胞RNA进行反转录,以β-actin作为内参,利用实时定量PCR方法,检测人群中FHL2基因相对表达水平,并分析其差异。结果：FHL2和β-actin基因的实时定量PCR熔解曲线均为单峰,所得到的Ct值与相应的PCR产物呈良好的线性关系;20例血液标本中FHL2表达水平之间存在较大差异,且其表达水平与性别无关。结论：公认的放射诱导基因FHL2可能不适合作为辐射生物剂量计。     

Reference Gene Selection for Normalization of PCR Analysis in Chicken Embryo Fibroblast Infected with H6N1 AIV

Chicken embryo fibroblasts(CEFs)are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus(AIV).In this study,the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR(QPCR)analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system.CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3,12,24 and 30 hours post-infection.The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR.Based on expression stability and expression levels,our data suggest that the ribosomal protein L4(RPL4)and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide(YWHAZ)are the best reference genes to use in the study of host cell response to H5N1 AIV infection.However,for the study of replication levels of H5N1 AIV in CEFs,the β-actin gene(ACTB)and the ribosomal protein L4(RPL4)gene are the best references.     

Comparison of in vitro and in vivo reference genes for internal standardization of real-time PCR data

Real-time PCR is a powerful technique for gene expression studies, which have become increasingly important in a large number of clinical and scientific fields. The significance of the obtained results strongly depends on the normalization of the data to compensate for differences between the samples. The most widely used approach is to use endogenous reference genes (housekeeping genes) as internal standards. This approach is controversially discussed in the literature because none of the reference genes is stably expressed throughout all biological samples. Therefore, candidate reference genes have to be validated for each experimental condition. In our studies, we introduced and evaluated an in vitro synthesized reference cRNA for internal standardization of relative messenger RNA (mRNA) expression patterns. This reference, consisting of the in vitro transcribed coding sequence of aequorin, a jellyfish protein, was incorporated in the extracted RNA. The experimental significance of this approach was representatively tested for the expression of the neurotrophin-3 mRNA in distinct regions of mouse brains. A comparison to three stably expressed reference genes [beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and hypoxanthine phosphoribosyl-transferase 1 (HPRT1)] gave evidence that the spiking of template RNA with in vitro transcribed cRNA is a valuable tool for internal standardization of real-time PCR experiments.     

基因表达转录分析中内参基因的选择

目前基因表达的转录分析多采用单一看家基因作为内参来校正目标基因的表达量.实验中以人肝癌BEL-7402细胞为研究对象,应用实时荧光定量PCR技术,观察了新型三肽化合物酪丝缬肽作用后RPL13A、UBC、EIF4A、B2M、GAPDH和ACTB共6个看家基因mRNA水平的表达情况.经过geNorm程序统计学分析处理,结果表明,这6个看家基因的表达存在差异,确定了RPL13A、UBC2个看家基因用于校正目标基因的表达量.基因表达转录分析中内参基因选择的必要性在实验中得以证明,更重要的是为各种实验因素影响下(尤其是新物质作用下)内参基因的选择介绍和提供了一种行之有效的方法.