Tissue-specific gene expression in soybean (Glycine max) detected by cDNA microarray analysis

We have constructed cDNA microarrays for soybean (Glycine maxL. Merrill), containing approximately 4,100 Unigene ESTs derived from axenic roots, to evaluate their application and utility for functional genomics of organ differentiation in legumes. We assessed microarray technology by conducting studies to evaluate the accuracy of microarray data and have found them to be both reliable and reproducible in repeat hybridisations. Several ESTs showed high levels (50 fold) of differential expression in either root or shoot tissue of soybean. A small number of physiologically interesting, and differentially expressed sequences found by microarray analysis were verified by both quantitative real-time RT-PCR and Northern blot analysis. There was a linear correlation (r² = 0.99, over 5 orders of magnitude) between microarray and quantitative real-time RT-PCR data. Microarray analysis of soybean has enormous potential not only for the discovery of new genes involved in tissue differentiation and function, but also to study the expression of previously characterised genes, gene networks and gene interactions in wild-type, mutant or transgenic plants.     

Neural progenitor genes. Germinal zone expression and analysis of genetic overlap in stem cell populations

The identification of the genes regulating neural progenitor cell (NPC) functions is of great importance to developmental neuroscience and neural repair. Previously, we combined genetic subtraction and microarray analysis to identify genes enriched in neural progenitor cultures. Here, we apply a strategy to further stratify the neural progenitor genes. In situ hybridization demonstrates expression in the central nervous system germinal zones of 54 clones so identified, making them highly relevant for study in brain and neural progenitor development. Using microarray analysis we find 73 genes enriched in three neural stem cell (NSC)-containing populations generated under different conditions. We use the custom microarray to identify 38 "stemness" genes, with enriched expression in the three NSC conditions and present in both embryonic stem cells and hematopoietic stem cells. However, comparison of expression profiles from these stem cell populations indicates that while there is shared gene expression, the amount of genetic overlap is no more than what would be expected by chance, indicating that different stem cells have largely different gene expression patterns. Taken together, these studies identify many genes not previously associated with neural progenitor cell biology and also provide a rational scheme for stratification of microarray data for functional analysis.     

UCA1, a non-protein-coding RNA up-regulated in bladder carcinoma and embryo, influencing cell growth and promoting invasion

A non-protein-coding RNA, UCA1, has been cloned from human bladder TCC cell line BLZ-211 by using 5' and 3' RACE. The UCA1 full-length cDNA was 1442 bp. RT-PCR analysis indicated that UCA1 is an embryonic development and bladder cancer-associated RNA. The proliferative, migrative, invasive, and drug resistance behaviors of human bladder TCC cell line BLS-211 were enhanced by exogenous UCA1 expression in vitro. Several potential target genes of UCA1 were identified through microarray analysis. Moreover, the expression of UCA1 also increased tumorigenic potential of BLS-211 cells in nude mice. Results from the present study suggested that UCA1 might play a pivotal role in bladder cancer progression and embryonic development.     

The gene expression profiling in murine cortical cells undergoing programmed cell death (PCD) induced by serum deprivation

PCD (programmed cell death) is important mechanism for development, homeostasis and disease. To analyze the gene expression pattern in brain cells undergoing PCD in response to serum deprivation, we analyzed the cDNA microarray consisting of 2,300 genes and 7 housekeeping genes of cortical cells derived from mouse embryonic brain. Cortical cells were induced apoptosis by serum deprivation for 8 hours. We identified 69 up-regulated genes and 21 down-regulated genes in apoptotic cells. Based on the cDNA microarray data four genes were selected and analyzed by RT-PCR and northern blotting. To characterize the role of UNC-51-like kinase (ULK2) gene in PCD, we investigated cell death effect by ULK2. And we examined expression of several genes that related with PCD. Especially GAPDH was increased by ULK2. Theses findings indicated that ULK2 is involved in apoptosis through p53 pathway.     

应用寡核苷酸芯片筛选维甲酸诱导神经母细胞瘤细胞系SH-SY5Y分化成神经元过程中的差异表达基因

目的:应用寡核苷酸芯片筛选维甲酸(RA)诱导神经母细胞瘤细胞系SH-SY5Y分化成神经元过程中的差异表达基因。方法:从人胎脑及不同类型神经系统肿瘤组织中获取目的基因,查询相应基因mRNA序列,设计并合成探针,制备了含218种基因的神经功能相关的寡核苷酸芯片。应用RA诱导SH-SY5Y8d分化成成熟神经元,提取对照组和实验组每天的总RNA,通过逆转录荧光标记cDNA探针并与芯片杂交,洗片后扫描获取图像,数据分析获得差异表达基因,并通过RT-PCR进行验证。结果:发现13种基因表达上调,没有得到下调基因。RT-PCR验证结果基本与芯片结果一致。结论:SH-SY5Y经RA诱导分化成神经元存在一些差异表达的基因,寡核苷酸芯片技术可为研究SH-SY5Y诱导分化成神经元的分子作用机理提供技术平台。     

Diagnostic plots for detecting outlying slides in a cDNA microarray experiment

Different sources of systematic and random error variations are often observed in cDNA microarray experiments. A simple scatter plot is commonly used to examine outlying slides that have unusual expression patterns or larger variability than other slides. These outlying slides tend to have large impacts on the subsequent analyses, such as identification of differentially expressed genes and clustering analysis. However, it is difficult to select outlying slides rigorously and consistently based on subjective human pattern recognition on their scatter plots. A graphical method and a rigorous diagnostic measure are proposed to detect outlying slides. The proposed graphical method is easy to implement and shown to be quite effective in detecting outlying slides in real microarray data sets. This diagnostic measure is also informative to compare variability among slides. Two cDNA microarray data sets are carefully examined to illustrate the proposed approach. A 3840-gene microarray experiment for neuronal differentiation of cortical stem cells and a 2076-gene microarray experiment for anticancer compound time-course expression of the NCI-60 cancer cell lines.     

Identification of molecules derived from human fibroblast feeder cells that support the proliferation of human embryonic stem cells

The majority of human embryonic stem cell lines depend on a feeder cell layer for continuous growth in vitro, so that they can remain in an undifferentiated state. Limited knowledge is available concerning the molecular mechanisms that underlie the capacity of feeder cells to support both the proliferation and pluripotency of these cells. Importantly, feeder cells generally lose their capacity to support human embryonic stem cell proliferation in vitro following long-term culture. In this study, we performed large-scale gene expression profiles of human foreskin fibroblasts during early, intermediate and late passages using a custom DNA microarray platform (NeuroStem 2.0 Chip). The microarray data was validated using RT-PCR and virtual SAGE analysis. Our comparative gene expression study identified a limited number of molecular targets potentially involved in the ability of human neonatal foreskin fibroblasts to serve as feeder cells for human embryonic stem cell cultures. Among these, the C-KIT, leptin and pigment epithelium-derived factor (PEDF) genes were the most interesting candidates.     

用基因芯片筛选前列腺癌LNCaP和C4-2细胞系中差异表达的基因

目的：筛选与前列腺癌进展相关的功能基因。方法：利用Affymetrix公司的人类金基因组U133A芯片,筛选在前列腺癌细胞系LNCaP和C4-2中差异表达的基因,并用RT-PCR方法验证部分差异基因的表达。结果：芯片筛选结果表明,与LNCaP细胞系相比,C4-2细胞系中217个基因转录本表达上调,101个基因转录本表达下调;对其中45个基因进行了RT-PCR验证,证明35个基因转录本的表达结果与芯片筛选一致。结论：筛选出了在LNCaP和C4-2细胞系中显著差异表达的基因35个,为进一步研究前列腺癌进展的机制奠定了基础。     

High Resolution Array-CGH Characterization of Human Stem Cells Using a Stem Cell Focused Microarray

Human embryonic and induced pluripotent stem cells (ESCs, iPSCs) that are cultured for an extended period of time are susceptible to genomic instability. Chromosomal aberrations decrease the reliability and reproducibility of experiments and could deem the cells unusable for therapeutic purposes. The genetic stability of human ESCs and iPSCs is commonly monitored by karyotype analysis. However, this low-resolution technique can only identify large aneuploidies. A reliable, high-resolution technique to detect genomic aberrations at a cost comparable to karyotyping is needed to better characterize stem cell lines. We have designed a stem cell focused array-comparative genomic hybridization microarray that covers the entire genome at high resolution with increased probe coverage in over 60 stem cell associated genes and more than 195 cancer related genes. Several iPSC lines were analyzed using the focused microarray and compared with either karyotyping or a standard Agilent 44K microarray. In addition to the abnormalities detected by these platforms, the custom microarray identified several small duplications spanning stem cell and/or cancer related genes. Scientists using a stem cell focused microarray to characterize their stem cells will be aware of the structural variants present in their cells and be more confident in their experimental results.