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。     

小桐子低温胁迫下microRNA实时荧光定量PCR内参的筛选与比较

选择合适的内参对实时荧光定量PCR(qRT-PCR)结果的准确性极其重要,在microRNA(miRNA)的qRT-PCR分析中尤为如此。通过筛选适宜分析小桐子低温胁迫下miRNA定量表达的内参,为小桐子及其他物种mi RNA的qRT-PCR分析提供有用的理论参考。基于以前的小RNA-seq结果,以低温处理的小桐子为材料,挑选11个候选内参基因,用实时荧光定量PCR技术检测它们在不同样本中的表达量,采用GeNorm、NormFinder和BestKeeper软件进行表达稳定性综合分析。结果表明,表达最稳定的基因是miR6448和U6,miR6448的Ct值为23左右,表达丰度适中;U6的Ct值为10左右,表达丰度高;因此,miR6448可作为小桐子低温胁迫下表达丰度适中的miRNA qRT-PCR的内参基因;U6可作为小桐子低温胁迫下丰度较高的miRNA qRT-PCR的内参基因。     

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。     

内参基因在昆虫实时荧光定量PCR中的研究进展

作为一种高效的定量PCR技术,实时荧光定量PCR(qRT-PCR)因其灵敏度高、特异性强、定量准确等优点,已被广泛运用于昆虫基因表达和转录分析。然而,为了控制样本RNA在质量和逆转录效率上存在差异,必须筛选表达稳定的"看家基因"作为内参基因,对目的基因表达量进行校正和标准化。许多学者研究表明,昆虫种类和实验条件的不同,导致选择的内参基因也不尽相同。因此,本文综述了前人有关昆虫内参基因的研究及其稳定性评价,为其它昆虫内参基因的研究提供理论参考依据。     

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。     

实时荧光定量PCR中内参基因的选择

实时荧光定量PCR技术是分析基因表达谱的一种常用方法,在分析中选择合适的内参基因对数据进行校正是得到可信数据的关键。以Lactobacillus helveticus H9为研究对象,应用实时荧光定量PCR技术,评价了5种常用内参基因ldh、recA、rpoB、gapdh和16S rRNA的表达稳定性,通过geNorm和NormFinder程序进行数据分析,结果表明5个候选内参基因在菌株不同的发酵时间点表达相对都较为稳定,结合两种分析得到其中最为稳定的基因是ldh,适合于用作后续实时荧光定量PCR试验中的内参基因。     

植物实时荧光定量PCR内参基因的特点及选择

实时荧光定量PCR(qRT-PCR)具有灵敏度高、特异性强、重复的动态定量范围和高通量等优点,是进行植物基因表达和转录分析最常用的技术手段之一.选择合适的内参基因是正确运用实时荧光定量PCR分析目标基因表达变化的前提.近年来,大量研究表明,内参基因的选择应取决于研究者的实验条件;随着实验条件的变化,内参基因的选择也随之变化.因此,实时荧光定量PCR结果分析的准确性在很大程度上依赖于所选择的内参基因是否适合.该文从内参基因的选择、常用内参基因的特点、新内参基因的挖掘、应用内参基因组合的优点和内参基因的稳定性评价等几方面进行综述,以期为研究者在实验中选择合适的内参基因提供参考和理论依据.     

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-tubulin 4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。     

藜科植物藜和灰绿藜实时荧光定量PCR内参基因的选择

选择合适的内参基因是实时荧光定量PCR(qRT-PCR)研究的关键,目前对藜科耐盐(盐生)植物胁迫相关基因的表达分析中所用内参基因的报道较为有限.该研究利用GeNorm、NormFinder和BestKeeper 3个内参基因分析软件,对已选择过的β-TUBULIN、β-ACTIN、GAPDH 3个常用候选内参基因进行了比较分析,筛选出在NaCl和PEG胁迫下藜和灰绿藜中表达相对稳定的内参基因.结果表明:GeNorm、NormFinder内参软件分析在NaCl和PEG胁迫下,GAPDH是藜和灰绿藜中均共同稳定表达的内参基因,同时在藜和灰绿藜中也有各自表达较稳定的内参基因,β-ACTIN在藜中稳定表达,β-TUBULIN则在灰绿藜中稳定表达.对相同科不同种的植物内参基因表达差异进行比较,内参基因在相同科中具有相同稳定表达的内参;对相同胁迫下两种不同植物内参基因表达稳定性进行分析,内参基因的选择需根据实际的实验材料和实验条件而定.基于3个分析软件对以上3个常用内参基因的分析结果,初步确定了在藜科植物藜和灰绿藜中相对稳定的内参基因,为藜和灰绿藜胁迫相关基因的定量表达分析提供了参考依据.     

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.     

Down-regulation of histone deacetylase 4, −5 and −6 as a mechanism of synergistic enhancement of apoptosis in human lung cancer cells treated with the combination of a synthetic retinoid,Am80 and green tea catechin

(−)-Epigallocatechin gallate (EGCG), a green tea catechin, acts as a synergist with various anticancer drugs, including retinoids. Am80 is a synthetic retinoid with a different structure from all-trans-retinoic acid: Am80 is now clinically utilized as a new drug for relapsed and intractable acute promyelocytic leukemia patients. Our experiments showed that the combination of EGCG and Am80 synergistically induced both apoptosis in human lung cancer cell line PC-9 and up-regulated expressions of growth arrest and DNA damage-inducible gene 153 (GADD153), death receptor 5, and p21^waf1 genes in the cells. To understand the mechanisms of synergistic anticancer activity of the combination, we gave special attention to the lysine acetylation of proteins. Proteomic analysis using nanoLC-ESI-MS/MS revealed that PC-9 cells treated with the combination contained 331 acetylated proteins, while nontreated cells contained 553 acetylated proteins, and 59 acetylated proteins were found in both groups. Among them, the combination increased acetylated-p53 and acetylated-α-tubulin through reduction of histone deacetylase (HDAC) activity in cytosol fraction, although the levels of acetylation in histones H3 or H4 did not change, and the combination reduced protein levels of HDAC4, −5 and −6 by 20% to 80%. Moreover, we found that a specific inhibitor of HDAC4 and −5 strongly induced p21^waf1 gene expression, and that of HDAC6 induced both GADD153 and p21^waf1 gene expression, which resulted in apoptosis. All results demonstrate that EGCG in combination with Am80 changes levels of acetylation in nonhistone proteins via down-regulation of HDAC4, −5 and −6 and stimulates apoptotic induction.     

Steroid degradation and two steroid-inducible enzymes in the marine bacterium H5

Natural and synthetic steroid hormones excreted into the environment are potentially threatening the population dynamics of all kinds of animals and public health. We have previously isolated a steroid degrading bacterial strain (H5) from the Baltic Sea, at Kiel, Germany. 16S-rRNA analysis showed that bacterial strain H5 belongs to the genus Vibrio, family Vibrionaceae and class Gamma-Proteobacteria. Bacterial strain H5 can degrade steroids such as testosterone and estrogens, which was shown in this study by determining the (3)H labeled steroid retaining in the bacterial H5 culture medium at incubation times of 5 h and 20 h. Since 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) is a key enzyme in adaptive steroid degradation in Comamonas testosteroni (C. testosteroni), in previous investigations, a meta-genomic system with the 3α-HSD/CR gene as a positive control was established. By this meta-genomic system, two estradiol inducible genes coding 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase, respectively, which are involved in steroid degradation, were found in marine strain H5. In the present work, the 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase genes were subcloned into plasmids pET38-12 and pET24-17, respectively. Overexpression in Escherichia coli (E. coli) strain BL21(DE3)pLysS cells resulted in corresponding proteins with an N-terminal His-tag sequence. After induction with isopropyl-β-D-thiogalactoside, 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase were purified in one step using nickel-chelate chromatography. After protein determination, 3-ketosteroid-delta-1-dehydrogenase (0.48 mg/ml) and carboxylesterase (1.28 mg/ml) were used to prepare antibodies to determine steroid binding specificity in future research. In summary, we have shown that the marine strain H5 could metabolize steroids; have isolated two estradiol inducible genes from strain H5 chromosomal DNA, and purified the corresponding proteins for further research. The exact characterization and systematic classification of the marine steroid degrading bacterial strain H5 is envisaged. The strain might be used for the bioremediation of steroid contaminations in seawater.     

Reduced O(2) concentration during CAM development - Its effect on angiogenesis and gene expression in the broiler embryo CAM

Hypoxia during embryogenesis may induce changes in the development of some physiological regulatory systems, thereby causing permanent phenotypic changes in the embryo. Various levels of hypoxia at different time points during embryogenesis were found to affect both anatomical and physiological morphogenesis. These changes and adaptations depended on the timing, intensity, and duration of the hypoxic exposure and, moreover, were regulated by differential expression of developmentally important genes, mostly expressed in a stage- and time-dependent manner. Eggs incubated in a 17%-oxygen atmosphere for 12h/d from E5 through E12 exhibited a clear and significant increase in the vascular area of the chorioallantoic membrane (CAM); an increase that was already significant within 12h after the end of the 1st hypoxic exposures (E6). We used the combination of the genes, β-actin, RPLP0 and HPRT as a reference for gene expression profiling, in studying the expression levels of hypoxia-inducible factor 1-alpha (HIF1α), vascular endothelial growth factor alpha-2 (VEGF α 2), vascular endothelial growth factor receptor 2 (KDR), matrix metalloproteinase-2 (MMP2), and fibroblast growth factor 2 (FGF2), under normal and hypoxic conditions. In general, expression of all five investigated genes throughout the embryonic day of development had similar patterns of hypoxia-induced alterations. In E5.5 embryos, expression of HIF1α, MMP2, VEGFα2, and KDR was significantly higher in hypoxic embryos than in controls. In E6 embryos expression of HIF1α, VEGFα2, and FGF2 was significantly higher in hypoxic embryos than in controls. From E6.5 onward expression levels of the examined genes did not show any differences between hypoxic and control embryos. It can be concluded that in this experimental model, exposing broiler embryos to 17% O(2) from E5 to E7 induced significant angiogenesis, as expressed by the above genes. Further studies to examine whether this early exposure to hypoxic condition affects the chick's ability to withstand a post-hatch hypoxic environment is still required.     

斑地锦RT-qPCR内参基因的筛选

实时荧光定量PCR(RT-qPCR)的前提条件之一是具有合适的内参基因。为筛选斑地锦(Euphorbia maculata)合适的RT-qPCR内参基因,该文利用同源克隆法克隆斑地锦GAPDH、EF-1α、act、UBQ、TUB-α、eIF-4A、CYP等基因片段,RT-qPCR检测7个候选内参基因在斑地锦不同生长期根、茎、叶和果实中的表达情况,并用geNorm、NormFinder和BestKeeper等生物学软件对各候选基因表达稳定性进行评价。结果表明:(1)克隆的GAPDH、EF-1α、act、UBQ、TUB-α、eIF-4A、CYP基因片段为729、808、753、422、233、656、313 bp,分别编码242、269、250、140、77、218、103个氨基酸,与其他植物相应氨基酸序列的最高同源性均在85%以上。(2)综合3个分析软件分析内参基因表达稳定性得出,表达稳定性排名为UBQ>EF-1α>TUB-α>eIF-4A>GAPDH>CYP>act。因此,可以选取UBQ作为斑地锦RT-qPCR分析的内参基因,用于不同生长期基因组织特异性表达研究。     

Species identification of Radix Astragali (Huangqi) by DNA sequence of its 5S-rRNA spacer domain

About 300 species and varieties of Astragalus are identified in China, making the identification of the origin of a particular Astragalus species on the consumer market difficult. A molecular genetic approach was developed to identify various species of Astragalus. Although the 5S-rRNA coding sequence is conserved in higher eukaryotes, the spacer domain of the 5S-rRNA gene has great diversity among different species. The 5S-rRNA spacer domain was amplified by polymerase chain reaction (PCR) from the isolated genomic DNA, and the PCR products (approximately 300 bp) covering the 5S-rRNA spacer domain were sequenced. The nucleotide sequences of Astragalus membranaceus, A. membranaceus var. mongholicus, A. lehmannianus, A. hoantchy, and of one closely related species Hedysarum polybotrys (Hongqi), were determined. Diversity in DNA sequence and restriction enzyme mapping among various species was found in their 5S-rRNA spacer domains. This is the first report on the detection of 5S-rRNA spacer region sequence of Astragalus, and the results could be used for genetic identification of Huangqi.