Profiling Expression Patterns and Isolating Differentially Expressed Genes by cDNA Microarray System with Colorimetry Detection

A high-density cDNA microarray with colorimetry detection system to simultaneously monitor the expression of many genes on nylon membrane is described and characterized. To quantify the expression of genes and to isolate differentially expressed genes, the southern hybridization process on filter membranes was employed. The levels of gene expression were represented by color intensities generated by colorimetric reactions in place of hazardous radioisotopes or costly laser-induced fluorescence detection. The gene expression patterns on nylon membranes were digitized by devices such as an economical flatbed scanner or a digital camera. The quantitative information of gene expression was retrieved by image analysis software. Quantitative comparison of the northern dot-blotting method with the microarray system is described. Applications employing single-color detection as well as dual-color detection to isolate differentially expressed genes among thousands of genes are demonstrated.     

用基因表达谱芯片研究人正常肝和肝细胞癌中差异表达的基因

以基因表达谱芯片对人正常肝及肝癌组织基因表达的差异性进行了研究比较。奖４０９６条人ｃＤＮＡ用点样仪点在特制玻片上制备成表达谱芯片;利用肝和肝癌组织的ｍＲＮＡ通过逆转录方法,将Ｃｙ３和Ｃｙ５２种荧光分别标记到两种组织的ｃＤＮＡ上,制备成ｃＤＮＡ探针,并与表达谱芯片进行杂交及扫描,重复４次实验,通过计算机数据处理判定基因是否在上述２种组织中有表达差异,筛选出差异表达的基因共９０３条。基因芯片技术可同时     

Identification of Differentially Expressed Genes in Distinct Skeletal Muscles in Cattle using cDNA Microarray

The 788-gene microarray was manufactured using selected elements from three different cDNA libraries in order to identify molecular processes that determine phenotypic characteristics between loin (M. longissimus thoracis) and round (M. semimembranosus) muscles. Microarray analyses identified 24 differentially expressed genes between the two muscles investigated. Five of the genes were verified by quantitative RT-PCR and three of them were mapped on bovine chromosomes using 5,000 rad bovine radiation hybrid (RH) panel. The map locations indicated that they were mapped in the same chromosomal regions where IMF and growth QTLs were located, suggesting that they are most possible positional candidate genes for the traits.     

Identification of Differentially Expressed Genes in the Pheromone Glands of Mated and Virgin Bombyx mori by Digital Gene Expression Profiling

Background

Mating decreases female receptivity and terminates sex pheromone production in moths. Although significant progress has been made in elucidating the mating-regulated inactivation of pheromone biosynthesis-activating neuropeptide (PBAN) secretion, little is known about the mating induced gene expression profiles in pheromone glands (PGs). In this study, the associated genes involved in Bombyx mori mating were identified through digital gene expression (DGE) profiling and subsequent RNA interference (RNAi) to elucidate the molecular mechanisms underlying the mating-regulated gene expression in PGs.

Results

Eight DGE libraries were constructed from the PGs of mated and virgin females: 1 h mating (M1)/virgin (V1) PGs, 3 h mating (M3)/virgin (V3) PGs, 24 h mating (M24)/virgin (V24) PGs and 48 h mating (M48)/virgin (V48) PGs (M48 and V48). These libraries were used to investigate the gene expression profiles affected by mating. DGE profiling revealed a series of genes showing differential expression in each set of mated and virgin female samples, including immune-associated genes, sex pheromone synthesis-associated genes, juvenile hormone (JH) signal-associated genes, etc. Most interestingly, JH signal was found to be activated by mating. Application of the JH mimics, methoprene to the newly-emerged virgin females leaded to the significant reduction of sex pheromone production. RNAi-mediated knockdown of putative JH receptor gene, Methoprene tolerant 1 (Met1), in female pupa resulted in a significant decrease in sex pheromone production in mature females, suggesting the importance of JH in sex pheromone synthesis.

Conclusion

A series of differentially expressed genes in PGs in response to mating was identified. This study improves our understanding of the role of JH signaling on the mating-elicited termination of sex pheromone production.     

Identification of Novel Genes Differentially Expressed in PMA-induced HL-60 Cells Using cDNA Microarrays

Identification of normal growth and differentiation-inducing proteins and their interaction in normal development have made it possible to elucidate the molecular basis of normal development and the mechanisms uncoupling growth and differentiation during tumor development. The development of cancer and the experimental reversal of tumorigenicity are accompanied by complex changes in patterns of gene expression. cDNA microarrays provide a powerful tool for studying these phenomena. In the present study, a high-density microarray of human cDNA elements was used to search for differences in gene expression associated with differentiation of human promyelic leukemia HL-60 cells. Microarrays containing 3,063 human cDNAs were printed on glass slides with high-speed robotics. These DNA chips were used to quantitatively monitor differential expression of the cognate human genes using a highly sensitive two-color hybridization assay. The identification of known and novel phorbol ester-regulated genes in hematopoietic progenitor cells demonstrates the sensitivity of the assay.     

Identification of Novel Genes Differentially Expressed in PMA-induced HL-60 Cells Using cDNA Microarrays

Identification of normal growth and differentiation-inducing proteins and their interaction in normal development have made it possible to elucidate the molecular basis of normal development and the mechanisms uncoupling growth and differentiation during tumor development. The development of cancer and the experimental reversal of tumorigenicity are accompanied by complex changes in patterns of gene expression. cDNA microarrays provide a powerful tool for studying these phenomena. In the present study, a high-density microarray of human cDNA elements was used to search for differences in gene expression associated with differentiation of human promyelic leukemia HL-60 cells. Microarrays containing 3,063 human cDNAs were printed on glass slides with high-speed robotics. These DNA 'chips' were used to quantitatively monitor differential expression of the cognate human genes using a highly sensitive two-color hybridization assay. The identification of known and novel phorbol ester-regulated genes in hematopoietic progenitor cells demonstrates the sensitivity of the assay.     

cDNA微阵列技术研究干旱胁迫下柽柳基因的表达

运用cDNA微阵列技术研究干旱胁迫下柽柳（Tamarix androssowii）基因的表达。分别将Cy5和Cy3两种荧光染料标记在干旱处理和对照的柽柳cDNA上,并与载有柽柳基因的微阵列进行杂交,通过计算机对芯片进行扫描和分析研究干旱胁迫下基因的表达。共获得了47个下调表达和62个上调表达的基因。Blastx分析表明这些基因按功能可以分为脱水保护、信号传导与调控、活性氧清除、光合作用、代谢、核糖体蛋白、蛋白质的分解与再生等几大类别。同时,发现了一些与干旱胁迫相关的功能未知基因和新基因。从而揭示了柽柳具有活性氧消除、代谢调节、脱水保护、蛋白质降解与再生等抗旱途径,并阐述了干旱胁迫前后柽柳基因的差异表达。     

用基因芯片结合荧光差异显示—PCR筛选和识别胃腺癌转移相关的基因

使用来源于同一个胃腺癌病人的原发灶RF－1（ATCC编号：CRL－1864）和转移灶RF－48细胞系（ATCC编号,CRL－1863）作为研究肿瘤转移分子机制的模型,RF－1（实验组）和RF－48（对照组）的mRNA通过逆转录方法,将Cy3和Cy5两种荧光染料分别标记到两种细胞的cDNA上,制备成cDNA探针,并与表达谱芯片（双点4096条基因）进行杂交与扫描,重复2次实验,利用计算机数据处理判断基因是否在上述两种细胞中有表达差异,共筛选出差异表达的基因共138条,其中81条在RF－48细胞中表达明显上调,57条在RF－48细胞中表达显著下调,同时也通过荧光差异显示－PCR（FDD－PCR）技术,克服了45个涉及胃腺癌转移相关基因,包括未被发现的基因3个,在两种筛选方法中都存在差异表达的基因共有7条,对部分可能与肿瘤志移机制有关的差异表达基因的作用进行了分析和讨论,基因芯片技术可高通量,大规模地研究基因表达水平,FDD－PCR技术可克隆出未发现的新基因,二者结合,初步筛选出与转移相关的基因,有助于揭示胃腺癌转移的分子机制。     

Powers of Multiple-Testing Procedures for Identification of Genes Significantly Differentially Expressed in Microarray Experiments

Because of the high operation costs involved in microarray experiments, the determination of the number of replicates required to detect a gene significantly differentially expressed in a given multiple-testing procedure is of considerable significance. Calculation of power/replicate numbers required in multiple-testing procedures provides design guidance for microarray experiments. Based on this model and by choice of a multiple-testing procedure, expression noises based on permutation resampling can be considerably minimized. The method for mixture distribution model is suitable to various microarray data types obtained from single noise sources, or from multiple noise sources. By using the biological replicate number required in microarray experiments for a given power or by determining the power required to detect a gene significantly differentially expressed, given the sample size, or the best multiple-testing method can be chosen. As an example, a single-distribution model of t-statistic was fitted to an observed microarray dataset of 3 000 genes responsive to stroke in rat, and then used to calculate powers of four popular multiple-testing procedures to detect a gene of an expression change D. The results show that the B-procedure had the lowest power to detect a gene of small change among the multiple-testing procedures, whereas the BH-procedure had the highest power. However, all multiple-testing procedures had the same power to identify a gene having the largest change. Similar to a single test, the power of the BH-procedure to detect a small change does not vary as the number of genes increases, but powers of the other three multiple-testing procedures decline as the number of genes increases.     

多重检验法在基因芯片研究中鉴定差异表达基因的统计功效

鉴于基因芯片实验的造价,在基因芯片实验设计中,首要考虑的因素是需要多少重复才能检测出一个具有显著差异表达的基因。计算多重检验法要求的重复数（样本大小）或功效可为基因芯片实验设计提供重要的参考。为此,本文基于置换重抽样法构建了一种基因表达噪声混合分布模型。该方法适用各类基因表达数据,即无论是基因表达单噪声源或是多噪声源都可行。应用混合模型和多重检验法并给定统计功效。研究者能在基因芯片实验中获得所需要的最少生物学重复数：或者根据样本大小来确定测定一个显著差异表达的基因所具有的检验功效;或者根据样本大小和统计检验功效,选择最好的统计测验方法。本文以一组在老鼠中与中风有关的3000个基因的基因芯片实验所获得的数据为例,应用该方法拟和后组建了一个单分布模型（即表达单噪声源的分布模型）。根据该模型,我们计算了4种多重检验法在鉴定一个具有表达差异（D）值的基因中所需要的统计功效。结果表明。检测一个小的差异D值,4种多重检验法中B方法的统计功效最低,而BH方法最高。但是,对于鉴定一个具有最大表达差异的基因时,4种方法有相同的鉴定功效。与传统的单个检验法一样,BH方法检测一个小的变化所需要的效率不会随基因数目增加而改变,其他3种多重检验法的检测功效则随基因数目增加而降低。     

Oligonucleotide Microarray Identifies Genes Differentially Expressed during Tumorigenesis of DMBA-Induced Pancreatic Cancer in Rats

The extremely dismal prognosis of pancreatic cancer (PC) is attributed, at least in part, to lack of early diagnosis. Therefore, identifying differentially expressed genes in multiple steps of tumorigenesis of PC is of great interest. In the present study, a 7,12-dimethylbenzanthraene (DMBA)-induced PC model was established in male Sprague-Dawley rats. The gene expression profile was screened using an oligonucleotide microarray, followed by real-time quantitative polymerase chain reaction (qRT-PCR) and immunohistochemical staining validation. A total of 661 differentially expressed genes were identified in stages of pancreatic carcinogenesis. According to GO classification, these genes were involved in multiple molecular pathways. Using two-way hierarchical clustering analysis, normal pancreas, acute and chronic pancreatitis, PanIN, early and advanced pancreatic cancer were completely discriminated. Furthermore, 11 upregulated and 142 downregulated genes (probes) were found by Mann-Kendall trend Monotone test, indicating homologous genes of rat and human. The qRT-PCR and immunohistochemistry analysis of CXCR7 and UBe2c, two of the identified genes, confirmed the microarray results. In human PC cell lines, knockdown of CXCR7 resulted in decreased migration and invasion. Collectively, our data identified several promising markers and therapeutic targets of PC based on a comprehensive screening and systemic validation.     

Discovery of Phytophthora infestans Genes Expressed in Planta through Mining of cDNA Libraries

Background

Phytophthora infestans (Mont.) de Bary causes late blight of potato and tomato, and has a broad host range within the Solanaceae family. Most studies of the Phytophthora – Solanum pathosystem have focused on gene expression in the host and have not analyzed pathogen gene expression in planta.

Methodology/Principal Findings

We describe in detail an in silico approach to mine ESTs from inoculated host plants deposited in a database in order to identify particular pathogen sequences associated with disease. We identified candidate effector genes through mining of 22,795 ESTs corresponding to P. infestans cDNA libraries in compatible and incompatible interactions with hosts from the Solanaceae family.

Conclusions/Significance

We annotated genes of P. infestans expressed in planta associated with late blight using different approaches and assigned putative functions to 373 out of the 501 sequences found in the P. infestans genome draft, including putative secreted proteins, domains associated with pathogenicity and poorly characterized proteins ideal for further experimental studies. Our study provides a methodology for analyzing cDNA libraries and provides an understanding of the plant – oomycete pathosystems that is independent of the host, condition, or type of sample by identifying genes of the pathogen expressed in planta.     

Estimation of the Proportion of Differentially Expressed Genes Using Hellinger Distance

We consider the problem of estimating the proportion of differentially expressed genes from the distribution of P-values arising from statistical tests in a microarray experiment. A two-component mixture model is studied in which one component is uniform on [0,1] and corresponds to equally expressed genes and the other component corresponds to differentially expressed genes. Two different parametric families are explicitly investigated for modeling the nonuniform component—the Beta family and a mixture of uniform densities whose supports form a partition of [0,1]. The Minimum Hellinger discrepancy is used to estimate the mixture proportions which are then used to estimate the proportion of differentially expressed genes in the microarray study. The performance of the proposed methods are investigated using simulation studies in which they are also compared with selected other published procedures. The methods are illustrated through a case study involving data from a published microarray experiment.