茶树实时荧光定量PCR分析中内参基因的选择

选择合适的内参基因是提高实时荧光定量PCR分析（qRT-PCR）准确性的先决条件。该文以茶树（Camellia sinensis）芽、叶、幼根、嫩茎、花瓣、种子和愈伤组织为材料,应用实时荧光定量PCR技术,分析了18S rRNA、GAPDH、β-actin和α-tubulin4个常用内参基因在茶树不同器官组织中的表达情况。经GeNorm和NormFinder软件分析发现,当利用荧光定量PCR分析比较茶树不同器官组织中的基因表达差异时,可选择β-actin作为校正内参基因;而比较不同成熟度的叶片和愈伤组织时,可以选择GAPDH作为校正内参基因。     

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     

岩白菜实时荧光定量PCR分析的内参基因的筛选

本研究为筛选在岩白菜中稳定表达的内参基因,以用于岩白菜实时荧光定量PCR研究,以岩白菜的幼叶、成熟叶、叶柄、根、一年生根状茎和二年生根状茎为材料,采用实时荧光定量PCR技术比较了GAPDH、18S rRNA、β-actin、TUA、UBQ5和TUB等6个候选内参基因的表达情况,并经Ge Norm、Norm Finder和Best Keeper等3种软件分析,分析表明6个候选内参基因在岩白菜不同器官中的表达情况存在差异,其中GAPDH和18S rRNA的表达都较为稳定,可作为岩白菜实时荧光定量PCR分析的内参基因。     

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.     

RNA质量对内参基因选择和qRT-PCR结果评价的影响

目的:研究RNA质量对内参基因选择和实时定量PCR(qRT-PCR)分析肿瘤坏死因子α(TNFα)诱导基因表达结果评价的影响。方法:用TNFα刺激体外培养的小鼠血管内皮细胞,采用qRT-PCR技术,以2种常用的管家基因β-actin和GAPDH为内参,检测细胞间黏附分子1(ICAM-1)基因的表达,探讨RNA纯度对TNFα下游基因表达检测结果的影响。结果:当提取的总RNA纯度很高(D260nm/D230nm2.0)时,以β-actin和GAPDH为内参基因检测ICAM-1基因表达的结果相近;当总RNA可能被盐及小分子污染(D260nm/D230nm2.0)时,以β-actin为内参基因检测的ICAM-1基因相对表达量显著高于以GAPDH为内参所得结果。结论:RNA质量显著影响荧光定量PCR对基因表达的检测结果,因此在RNA质量较低时应选择2个或以上内参基因进行校正,以减少实验结果误差,得到准确结果。     

水曲柳花发育过程中AG、SOC1基因表达的qRT-PCR分析

以水曲柳(Fraxinus mandshurica Rupr.)不同时期的雌雄花为材料,应用实时荧光定量PCR技术,分析了18S rRNA、GAPDH、β-actin和TU 4个常用内参基因在水曲柳花中的表达情况。经NormFinder软件分析选择出TU作为内参基因,并对水曲柳中的开花相关基因AG和SOC1在开花不同时期的表达情况进行了分析,结果表明：在水曲柳花发育期间AG、SOC1表达量在雌雄花间存在差异,AG基因在雌花初期表达量最高,之后逐渐降低,到了成熟胚囊时AG表达量又有所增加;而在雄花中AG在减数分裂时最高为1.53。SOC1基因在雌花中的表达量低于雄花。这一研究结果为深入了解水曲柳花发育过程中相关基因的表达提供理论依据。     

光处理下粘虫头部组织定量PCR分析中内参基因的筛选

[目的]筛选不同光处理下粘虫Mythimna separata稳定表达的内参基因,为基因定量研究提供基础.[方法]以粘虫头部组织为材料,选取18s rRNA、EF-1α、β-actin、GAPDH和AK5种候选内参基因进行实时荧光定量PCR(qRT-PCR)分析,然后通过△Ct法,BestKeeper、GeNorrn、Normfinder和RefFinder软件对候选内参基因的稳定性进行分析;利用GeNorm软件进行基因配对差异分析,以判断内参基因的最适组合.[结果]5种候选内参基因的Ct值都处于15-28之间.4种软件对5个候选基因稳定性的分析结果存在一定差异.综合分析各种软件的分析结果,推荐粘虫成虫不同光处理条件下采用AK和GAPDH作为内参基因.[结论]根据特定试验体系选择合适的内参基因对于qRT-PCR定量结果的准确定和可靠性具有重要意义.本研究对后续粘虫在不同光处理条件下目标基因的准确定量具有重要意义.     

翘嘴鳜per1 mRNA表达量分析中采用的内参基因稳定性比较

为筛选生物钟核心基因per1表达定量中的相对稳定性最好的内参基因,本研究取翘嘴鳜成鱼心脏、肝脏、肾脏、脑、红肌、白肌、肠、眼和脾等九个组织为研究对象,选取GAPDH、18S rRNA、β-actin、rps29、RPL13a、B2M和EF1a为内参基因,采用实时荧光定量PCR(qRT-PCR)对per1基因mRNA表达水平进行检测分析。研究结果表明18S rRNA和GAPDH的平均稳定值M最低,相对表达量最稳定。以18S rRNA和GAPDH为内参基因时分析发现per1基因表达量在肝脏中最高。本研究为在鱼类per1 mRNA表达检测过程中选用稳定的内参基因提供了实验和理论参考。     

实时荧光定量PCR分析中毛果杨内参基因的筛选和验证

实时荧光定量PCR(qRT-PCR)技术具有高灵敏性、高保真性和高特异性, 被广泛应用于基因表达的分析。在数据处理过程中, 选用稳定表达的基因作为内参基因对准确分析实验结果非常关键。以毛果杨(Populus trichocarpa)的不同组织以及锌胁迫下的组培苗为材料, 使用荧光定量PCR方法分析了TUA8、TUB6、ubiquitin、GAPDH、actin、18S rRNA和EF1α 7个看家基因的表达情况。通过geNorm、NormFinder和BestKeeper 3个程序的综合分析, 发现actin、ubiquitin、EF1α和18S rRNA的稳定性较好, 可用作毛果杨基因表达研究的内参基因; 而TUB6在不同组织中稳定性最差; GAPDH在锌胁迫下的组织中稳定性最差, 因此不适宜作为内参基因。毛果杨NAC基因的表达分析, 进一步验证了上述结果。该研究对采用qRT-PCR方法分析毛果杨基因表达过程中内参基因的选择具有指导作用, 同时对揭示NAC基因的功能也有一定的意义。     

山羊不同组织器官的内参基因筛选

本研究通过比较9个内参基因在山羊不同组织中的表达水平进而确定最适合研究山羊组织表达的内参基因。本试验以简州大耳羊为试验材料,利用实时荧光定量PCR技术分析9个内参基因(GAPDH,PPIA,18S rRNA,PPIB,UXT,RPLP0,ACTB,EIF3K和TBP)在心脏、肝脏、脾脏、肺脏、肾脏、大肠、瘤胃、背最长肌和皮下脂肪等组织中的表达差异情况,并利用geNorm、NormFinder和BestKeeper等程序分析了它们的表达稳定性。geNorm和NormFinder程序一致显示TBP表达最稳定,其次是UXT和RPLP0;BestKeeper分析显示18S rRNA表达最为稳定,其次为TBP和ACTB;3个程序一致认为GAPDH表达稳定性最差。综合3个程序分析得出TBP最适合作为山羊组织中的内参基因,其次为UXT和RPLP0,GAPDH表达稳定性最差,不适合作为山羊组织内参,这为后续研究其他目的基因在山羊组织器官中的表达模式提供数据保障。     

Validation of Reference Genes for Quantitative Real-Time PCR in Bovine PBMCs Transformed and Non-transformed by Theileria annulata

Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (β-actin), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata.     

A Panel of Stably Expressed Reference Genes for Real-Time qPCR Gene Expression Studies of Mallards (Anas platyrhynchos)

Determining which reference genes have the highest stability, and are therefore appropriate for normalising data, is a crucial step in the design of real-time quantitative PCR (qPCR) gene expression studies. This is particularly warranted in non-model and ecologically important species for which appropriate reference genes are lacking, such as the mallard—a key reservoir of many diseases with relevance for human and livestock health. Previous studies assessing gene expression changes as a consequence of infection in mallards have nearly universally used β-actin and/or GAPDH as reference genes without confirming their suitability as normalisers. The use of reference genes at random, without regard for stability of expression across treatment groups, can result in erroneous interpretation of data. Here, eleven putative reference genes for use in gene expression studies of the mallard were evaluated, across six different tissues, using a low pathogenic avian influenza A virus infection model. Tissue type influenced the selection of reference genes, whereby different genes were stable in blood, spleen, lung, gastrointestinal tract and colon. β-actin and GAPDH generally displayed low stability and are therefore inappropriate reference genes in many cases. The use of different algorithms (GeNorm and NormFinder) affected stability rankings, but for both algorithms it was possible to find a combination of two stable reference genes with which to normalise qPCR data in mallards. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies in ducks. The fact that nearly all previous studies of the influence of pathogen infection on mallard gene expression have used a single, non-validated reference gene is problematic. The toolkit of putative reference genes provided here offers a solid foundation for future studies of gene expression in mallards and other waterfowl.     

五龄飞蝗不同发育时间实时定量PCR内参基因的筛选

【目的】筛选5龄飞蝗不同发育时间的最适内参基因,为相关研究提供基础数据。【方法】本文选取β-肌动蛋白(β-actin)、延长因子(EF-1α)、3-磷酸甘油醛脱氢酶(GAPDH)、核糖体蛋白49(RP49)、α-微管蛋白(α-Tubulin)和18S核糖体RNA(18S rRNA)基因作为候选内参基因,运用实时定量PCR(qPCR)方法研究各基因在5龄飞蝗不同发育时间的相对表达量,用geNorm与Normfmder软件分析这6个基因表达稳定性。【结果】geNorm分析结果显示6个内参基因表达稳定度M值顺序为:β-actin(0.3720)>RP49(0.3750)>α-Tubulin(0.4030)>18S rRNA(0.4270)>EF-1α(0.4970)>GAPDH(0.6040)。M值越小表示基因表达稳定度越高,同时geNorm软件以标准化因子配对差异值(Pairwise variations)0.15默认为取舍值,由于V2/3=0.098<0.15,所以最适内参基因数目为2个。运用NormFinder软件也得出相似的结果。【结论】β-actin与RP49为5龄飞蝗不同发育时间的最适内参基因。     

Evaluation of housekeeping genes as references for quantitative real-time PCR analysis of European eel,Anguilla anguilla

Eels are important aquaculture species for which an increasing number of reference genes are being identified and applied. In this study, five housekeeping genes [RPL7 (ribosomal protein L7), 18 S (18 S ribosomal RNA), EF1A (elongation factor 1α), ACTB (β-actin) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase)] were chosen to evaluate their reliability as reference genes for quantitative real-time PCR (qPCR) for the study of Anguilla anguilla. The expression of the selected genes in different eel tissues was determined using qPCR at different growth stages or upon challenge by Anguillid herpesvirus (AngHV), and the expression levels of these genes were then compared and evaluated using the geNorm and NormFinder algorithms. Then, RefFinder was used to comprehensively rank the examined housekeeping genes. Interestingly, the expression of the evaluated housekeeping genes exhibited tissue-dependent and treatment-dependent variations. In different growth periods A. anguilla tissues, the most stable genes were the following: ACTB in mucus; 18 S in skin and kidney; RPL7 in muscle, gill, intestine and brain; EF1A in heart and liver; and GAPDH in spleen. In contrast, in AngHV-challenged A. anguilla tissues, the most stable genes were the following: 18 S in mucus; RPL7 in skin, gill, heart, spleen, kidney and intestine; EF1A in muscle and liver; and ACTB in brain. Further comparison analysis indicated that the expression of RPL7 and EF1A was stable in multiple A. anguilla tissues in different growth periods and in eels challenged by AngHV. Nonetheless, the expression level of GAPDH in eel tissues was lower, and it was unstable in several tissues. These results indicated that the selection of reference genes for qPCR analysis in A. anguilla should be made in accordance with experimental parameters, and both RPL7 and EF1A could be used as reference genes for qPCR study of A. anguilla at different growth stages or upon challenge by AngHV. The reference genes identified in this study could improve the accuracy of qPCR data and facilitate further studies aimed at understanding the biology of eels.     

Selection of Suitable Reference Genes for Quantitative Real-Time PCR in Apoptosis-Induced MCF-7 Breast Cancer Cells

Apoptosis is induced in MCF-7 breast cancer cells following treatment with salicylic acid (20 mM), either in the presence or absence of a heat shock (42°C for 30 min). In order to study the alterations of apoptotic genes with quantitative real-time PCR (qPCR), suitable genes with unchanged expression following the treatments is required for normalizing the gene expression levels. In this study, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin (ACTB), Histone H2A (HIST), constitutively expressed heat shock protein 70 (HSC70) and tyrosine 3-monooxygenase/trytophan 5 monooxygenase activation protein, 14-3-3 (YWHAZ) were evaluated as appropriate reference genes. Analysis of gene expression data with one-way ANOVA, geNorm and NormFinder identified HIST and YWHAZ as the least affected during the induction of apoptosis by the different treatments, and is the most suitable gene-pair for normalization during qPCR analysis in MCF-7 breast cancer cells undergoing apoptosis following treatment with SA and/or HS.