Gene expression profile analysis in astaxanthin-induced Haematococcus pluvialis using a cDNA microarray

The unicellular green alga Haematococcus pluvialis (Volvocales) is known for the ketocarotenoid astaxanthin (3, 3′-dihydroxy-β, β-carotene-4, 4′-dione) accumulation, which is induced under unfavorable culture conditions. In this work, we used cDNA microarray analysis to screen differentially expressed genes in H. pluvialis under astaxanthin-inductive culture conditions, such as combination of cell exposure to high irradiance and nutrient deprivation. Among the 965 genes in the cDNA array, there are 144 genes exhibiting differential expression (twofold changes) under these conditions. A significant decrease in the expression of photosynthesis-related genes was shown in astaxanthin-accumulating cells (red cells). Defense- or stress-related genes and signal transduction genes were also induced in the red cells. A comparison of microarray and real-time PCR analysis showed good correlation between the differentially expressed genes by the two methods. Our results indicate that the cDNA microarray approach, as employed in this work, can be relied upon and used to monitor gene expression profiles in H. pluvialis. In addition, the genes that were differentially expressed during astaxanthin induction are suitable candidates for further study and can be used as tools for dissecting the molecular mechanism of this unique pigment accumulation process in the green alga H. pluvialis. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Gastrointestinal complications of mitochondrial disease

The clinical presentation of mitochondrial disease can involve a number of organ systems which are seemingly unrelated. Gastrointestinal manifestations are often an early presentation of mitochondrial disorders. Dysphagia, feeding difficulties, gastroesophageal reflux, dysmotility with delayed gastric emptying, intestinal pseudo-obstruction, diarrhea and pancreatic exocrine insufficiency are amongst the major complications. Neuromuscular dysfunction affecting peristalsis is a common pathophysiologic mechanism causing many of these symptoms and treatment is primarily supportive.     

Quantification of mitochondrial DNA copy number: Pre-analytical factors

Mitochondrial DNA (mtDNA) content is important for understanding many cellular processes. Several pre-analytical factors, from sample collection to DNA extraction can affect measurement of mtDNA copy number. In the present study, whole blood samples yielded a higher mtDNA copy number than buffy coat samples. mtDNA content is affected by the cell separation method used and the time between blood withdrawal and cell separation. Thus, reference values must be established with the same type of sample. As to the DNA isolation and purification method, the manual phenol method can give randomly false high values. The QIAamp DNA Mini Kit provided the most highly reproducible mtDNA/nDNA yield.     

Gene expression profiling of human ovarian epithelial tumors by digo nucleotide microarray

Gene expression of human ovarian carcinoma cell lines and epithelial ovarian tumors was examined by oligonucleotide microarray for about 6000 human cDNAs. (1) Comparison of gene expression between CDDP-sensitive human ovarian serous adenocarcinoma cell lines and CDDP-resistant cell lines revealed that gamma-glutamylcysteine synthetase, glutathione peroxidase-like protein, dehydrogenase (UGDH), NAD(P)H: quinoneoxireductase, glucose-6-phosphatase, ornithine decarboxylase and dihydrodiol dehydrogenase were associated with a mechanism of CDDP-resistance. Comparison of gene expression between taxol-sensitive human ovarian cell lines and taxol-resistant cell lines showed that up-regulation of 30 kinds of gene expression including MDR and semaphorin E in taxol-resistant cell lines. (2) Comparison of gene expression among serous adenocarcinomas, clear cell adenocarcinomas and non-cancerous ovarian tissues by hierarchical clustering demonstrated that clear difference between carcinomas and non-cancerous ovarian tissues but not obvious difference between serous and clear adenocarcinomas. Genes that were up- and down-regulated specifically in these two types of ovarian carcinomas were further selected by the criteria that difference in the mRNA level by more than 4-fold between tumors and non-cancerous tissues. Tissue type specific alterations of gene expression are likely to play important roles in the carcinogenesis of epithelial ovarian tumors. cDNA microarray is a powerful and high-throughput tool to analyze gene expression of cancer development.     

异色瓢虫多巴脱羧酶基因序列及低温诱导表达分析

多巴脱羧酶(dopa decarboxylase,DDC)是把多巴降解成多巴胺的重要酶类,在昆虫行为和发育中具有重要作用。本研究在转录组获得异色瓢虫Harmonia axyridis DDC基因序列的基础上,从ORF两端设计特异性引物进行扩增并测序验证,获得了DDC基因的c DNA的ORF全长序列,其包含1431 bp,编码476个氨基酸,软件分析预测显示该基因编码蛋白的分子量为53.88 k Da,理论等电点为5.80。本实验分为升温、降温、低温储存以及不同发育阶段(包括预蛹,1-3 d蛹,1-3 d 4龄幼虫以及羽化1-3 d成虫)4个处理组,采用荧光定量PCR技术研究异色瓢虫不同处理组DDC基因的表达水平。结果表明:DDC基因在蛹期第1天的表达量最高,在升温诱导条件下表达无差异,降温诱导下表达量上升;黄色雌成虫低温储存条件下基因表达量先显著上升后显著下降,黑色雌成虫表达量无差异,表明DDC基因可以在低温胁迫下高表达促使异色瓢虫适应环境变化。     

Absolute quantitation of a heteroplasmic mitochondrial DNA deletion using a multiplex three-primer real-time PCR assay

Quantitation of wild-type and deleted mitochondrial DNA (mtDNA) coexisting within the same cell (a.k.a., heteroplasmy) is important in mitochondrial disease and aging. We report the development of a multiplex three-primer PCR assay that is capable of absolute quantitation of wild-type and deleted mtDNA simultaneously. Molecular beacons were designed to hybridize with either type of mtDNA molecule, allowing real-time detection during PCR amplification. The assay is specific and can detect down to six copies of mtDNA, making it suitable for single-cell analyses. The relative standard deviation in the threshold cycle number is approximately 0.6%. Heteroplasmy was quantitated in individual cytoplasmic hybrid cells (cybrids), containing a large mtDNA deletion, and bulk cell samples. Individual cybrid cells contained 100-2600 copies of wild-type mtDNA and 950-4700 copies of deleted mtDNA, and the percentage of heteroplasmy ranged from 43+/-16 to 95+/-16%. The average amount of total mtDNA was 3800+/-1600 copies/cybrid cell, and the average percentage of heteroplasmy correlated well with the bulk cell sample. The single-cell analysis also revealed that heteroplasmy in individual cells is highly heterogeneous. This assay will be useful for monitoring clonal expansions of mtDNA deletions and investigating the role of heteroplasmy in cell-to-cell heterogeneity in cellular models of mitochondrial disease and aging.     

Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.     

Gene expression profiling of cardiovascular disease models

Recent development of gene expression profiling technologies has enabled the large-scale analysis of gene expression changes during disease progression. Frequently, cardiovascular diseases involve complex interactions of multiple cell types over prolonged periods of time. A better understanding of the pathology of cardiovascular diseases and the potential identification of underlying genetic defects are currently being explored by using profiling methodologies in a number of animal and tissue-culture models.     

鸡3种组织中热应激相关基因的表达谱芯片分析

文章以8日龄矮小型黄羽肉雏鸡为材料,以28℃±1℃处理3 h为对照组,40℃±1℃热应激3 h为实验组,采集大脑、肝脏、腿肌3种组织,利用全基因组表达谱芯片检测实验组和对照组各组织的差异表达基因,并结合生物信息学方法分析其功能。研究结果表明：在热应激处理组与对照组中,大脑组织中共有183个显著变化的差异表达探针(变化倍数≥2或≤0.5,q≤0.05),129个有基因注释,39个生物学过程发生了改变,18个生物学通路发生显著改变;肝脏组织中有558个显著改变的探针(变化倍数≥2或≤0.5,q≤0.05),包含339个基因,涉及37个生物学过程,58个生物学通路发生改变;腿肌组织中显著改变的探针有301个(变化倍数≥2或≤0.5,q≤0.05),209个有基因注释,参与了31个生物学过程,51个生物学通路发生改变。3种组织中,获得差异表达探针935个,不同组织具有特异性的表达模式;多种热休克蛋白家族基因被诱导上调表达,抗凋亡过程、钙离子信号通路、MAPK信号通路和多种代谢途径发生改变。文章对鸡的热应激分子机制进行了初步探索,为筛选耐热性相关基因提供了理论依据。     

游离线粒体拷贝数检测方法建立及应用

摘要 目的：线粒体在生理和病理过程中都起着重要作用,线粒体破碎后形成的游离线粒体与一系列疾病密切相关。然而,人体内游离线粒体的含量较低很难被稳定抽提且易降解等因素导致游离线粒体拷贝数检测具有极大挑战。本研究拟建立一种快速、准确检测外周血游离线粒体拷贝数定量PCR技术。方法：通过多重荧光定量PCR技术在SLAN?-96S全自动医用PCR分析系统上检测人外周血游离线粒体拷贝数,构建新的游离线粒体检测方案。游离核基因在人体外周血中的稳定性远大于游离线粒体,因此使用多拷贝参考基因YH-1（300拷贝）检测游离核基因作为对照组。结果：成功建立了核基因标准曲线和线粒体标准曲线,并筛选游离线粒体拷贝数检测最佳引物扩增片段长度为82bp、血清有效分离时间在2h内、血清最佳分离方案为1600 r/min 离心10 min再16000 r/min 离心10 min、磁珠法游离核酸抽提试剂盒抽提游离核酸得率最高的新流程。利用新方案对100 例不同年龄段的随机人群外周血抽提游离线粒体拷贝数进行检测,结果显示30-79岁游离线粒体拷贝数与年龄之间的相关性参数为｜R｜= 0.18、P value = 0.077,游离线粒体拷贝数与性别之间的相关性参数为｜R｜= 0.27、P value = 0.061即游离线粒体拷贝数与年龄和性别均无显著相关性,研究结果与报道一致。结论：表明优化后的方案可稳定检测游离线粒体拷贝数,提供了一种快速、准确检测游离线粒体拷贝数的方法。     

Gene mining: a novel and powerful ensemble decision approach to hunting for disease genes using microarray expression profiling

Current applications of microarrays focus on precise classification or discovery of biological types, for example tumor versus normal phenotypes in cancer research. Several challenging scientific tasks in the post-genomic epoch, like hunting for the genes underlying complex diseases from genome-wide gene expression profiles and thereby building the corresponding gene networks, are largely overlooked because of the lack of an efficient analysis approach. We have thus developed an innovative ensemble decision approach, which can efficiently perform multiple gene mining tasks. An application of this approach to analyze two publicly available data sets (colon data and leukemia data) identified 20 highly significant colon cancer genes and 23 highly significant molecular signatures for refining the acute leukemia phenotype, most of which have been verified either by biological experiments or by alternative analysis approaches. Furthermore, the globally optimal gene subsets identified by the novel approach have so far achieved the highest accuracy for classification of colon cancer tissue types. Establishment of this analysis strategy has offered the promise of advancing microarray technology as a means of deciphering the involved genetic complexities of complex diseases.     

SSH 法结合定量 PCR 技术研究双肌臀猪 肌肉组织的差异表达基因

利用抑制性消减杂交技术 (suppression subtractive hybridization , SSH) 成功地构建了双肌臀与非双肌臀大白猪肌肉组织差异表达的消减 cDNA 文库,获得有效克隆 686 个. 对整个文库测序分析,共获得 587 条有效序列. 利用 BLAST 在线软件与 GenBank、 GenBank EST 和 Tigr Porcine EST 等数据库进行同源序列比较,发现其中有 11 个未知新序列,可能代表“双肌臀”大白猪肌肉组织特异表达的新基因. 对文库中所包含的兰尼啶受体基因 (RYR1)、钙依赖性蛋白激酶 基因 (CAMK2)、人类胰岛素生长因子结合蛋白 -7 基因 (IGFBP7),以及 695号、 882号和480号3个新基因进行了实时荧光定量 PCR 检测,结果显示： RYR1、 CAMK2 及 IGFBP7 基因在双肌臀猪肌肉组织 RNA 池中的表达量,分别为对照的非双肌臀猪肌肉组织 RNA 池中表达量的 1.87、 1.90 和 1.85 倍; 695 号、 882 号和 480 号 3 个新的 ESTs 在双肌臀猪肌肉组织 RNA 池中的表达量为非双肌臀猪肌肉组织 RNA 池中表达量的 1.48、 1.44 和 1.78 倍. 这提示我们,上述基因的上调表达很可能与猪双肌臀性状的发生有密切的关系,可以作为研究该性状的候选基因.     

Gene therapy for mitochondrial disease by delivering restriction endonucleaseSmaI into mitochondria

The restriction endonucleaseSmaI has been used for the diagnosis of neurogenic muscle weakness, ataxia and retinitis pigmentosa disease or Leigh's disease, caused by the Mt8993TG mutation which results in a Leu156Arg replacement that blocks proton translocation activity of subunit a of F₀F₁-ATPase. Our ultimate goal is to applySmaI to gene therapy for this disease, because the mutant mitochondrial DNA (mtDNA) coexists with the wild-type mtDNA (heteroplasmy), and because only the mutant mtDNA, but not the wild-type mtDNA, is selectively restricted by the enzyme. For this purpose, we transiently expressed theSmaI gene fused to a mitochondrial targeting sequence in cybrids carrying the mutant mtDNA. Here, we demonstrate that mitochondria targeted by theSmaI enzyme showed specific elimination of the mutant mtDNA. This elimination was followed with repopulation by the wild-type mtDNA, resulting in restoration of both the normal intracellular ATP level and normal mitochondrial membrane potential. Furthermore, in vivo electroporation of the plasmids expressing mitochondrion-targetedEcoRI induced a decrease in cytochromec oxidase activity in hamster skeletal muscles while causing no degenerative changes in nuclei. Delivery of restriction enzymes into mitochondria is a novel strategy for gene therapy of a special form of mitochondrial diseases.     

红系特异的GFP基因在转基因小鼠中的整合和表达

应用荧光定量PCR技术对由位点控制区LCR的HS2元件和 β 珠蛋白基因启动子指导的红系特异表达绿色荧光蛋白 (GFP)基因的转基因小鼠中外源基因拷贝数进行测定 ,使用荧光显微镜和流式细胞仪检测小鼠外周血中GFP的表达水平 ,并运用荧光原位杂交技术 (FISH)确定了其中两只转基因小鼠中外源基因的整合位点 ,结果表明 :在转基因小鼠中外源基因的拷贝数各不相同且相差较大 ,而且拷贝数与GFP基因的表达量之间未呈现出相关性 ;FISH分析确定出两只转基因小鼠的外源基因整合于不同的染色体上 ;杂交信号的强弱与拷贝数的多少相一致     

实时荧光定量PCR在固氮酶基因检测中的应用

实时荧光定量PCR是近年发展起来的一种新的实时定量检测特定核酸技术,它是核酸探针技术、荧光共振能量传递技术和PCR技术的有机结合。与常规PCR相比,它具有特异性更强、能有效解决PCR污染问题、自动化程度高等特点,扩大了PCR的应用范围。概述实时荧光定量PCR技术在固氮酶(nifH)基因检测中的应用与研究进展,并探讨该技术的发展和应用前景。     

黄龙病菌侵染过程中柑橘CsMYB基因克隆及表达分析

MYB类蛋白是一类与植物防卫反应有关的转录因子家族,本研究利用构建的甜橙健株与感染黄龙病的病株差减（SSH）文库,采用RACE技术克隆了一个MYB类基因的cDNA全长序列,命名为CsMYB（GenBank登录号为HQ841074）。柑橘CsMYB基因的cDNA全长为1 306 bp,生物信息学分析显示,该基因包括一个909 bp的完整开放读码框以及一个典型的26 bp poly-A,编码302个氨基酸,分子量为32.97 kD,等电点为8.5。同时,还有MYB类基因的保守特征区域,即在N端有两个典型的MYB DNA结合域：R2和R3。实时荧光定量PCR分析表明,柑橘CsMYB基因受到黄龙病菌侵染后的不同时期表达量不同,伴随着黄龙病病程的变化呈现不同的表达变化。推测CsMYB基因是一个转录因子,可能参与柑橘对黄龙病菌的防御反应过程。     

Gene expression profile of cytokines in patients with chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation with reduced conditioning

There are no reliable markers useful to predict the onset or the evolution of chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT), although several candidate biomarkers have been identified from limited hypothesis-driven studies. In this study we evaluated 14 patients who received a reduced intensity conditioning HSCT. Seven patients had cGVHD, whereas 7 never developed cGVHD during the period of observation. The expression of 114 cytokines in immunoselected cell populations was explored by microarray analysis and 11 cytokines were selected for further evaluation by real-time PCR. Differential gene expression measurements showed a significant up-regulation for INFγ (interferon, gamma) in CD8+ and for TNFSF3 (tumor necrosis factor superfamily, member 3) and for TNFSF10 (tumor necrosis factor superfamily, member 10) in CD14+ cell population when comparing cGVHD with control samples. The expression levels were significantly decreased for TNFSF10 in CD8+ cell population and for TNFSF12 (tumor necrosis factor superfamily, member 12) and for PDGFβ (platelet-derived growth factor, beta) in CD4+. Our data seem to suggest that different immune populations can play a role in cGVHD pathogenesis and the early detection of gene expression profile in these patients could be useful in the monitoring of GVHD. We hypothesized that PDGFβ down-regulation could represent a negative feedback to compensate for enhanced expression of its receptor recently reported.