Robust ordered mRNA differential display: an improved method for global gene expression profiling

Global gene expression profiling (GGEP) plays a pivotal role in biological research. We developed an improved GGEP method called "robust ordered mRNA differential display (RoDD)" by combining mRNA differential display (DD) and complementary DNA amplified fragment length polymorphisms (cDNA-AFLP) using elaborately designed primers and a poly (dT:A) replacement technique. Redundancy was minimized by bead-based isolation and coverage was improved by using restriction enzymes that recognized 4-bp sites. This method offers the common virtues of gel-based methods along with the reliability of cDNA-AFLP. The most significant advantage of RoDD over current gel-based methods is greatly improved coverage and minimized redundancy.     

基于荧光微球液相基因表达阵列的建立

建立基于荧光微球的液相基因表达阵列,用于特定基因组合的表达谱分析.采用带有不同强度荧光鉴别信号的羧基化微球,与氨基修饰的不同标签寡核苷酸序列化学偶联,制成微球阵列.多重连接依赖的探针扩增技术(MLPA)用于扩增靶基因核苷酸序列,即通过RNA标本六随机引物逆转成cDNA,与不同基因特异性的一对探针杂交,耐高温的连接酶联接,最后采用生物素标记的同一对引物扩增.PCR产物与微球阵列液相杂交,加入链亲和素标记的PE染料,上流式细胞仪检测.应用这一系统检测骨髓增生异常综合症中难治性贫血(RA)、难治性贫血伴原始细胞增多(RAEB)、难治性贫血伴转化中原始细胞增多(RAEBt)、急性髓细胞性白血病(AML)和其他组(包括再生障碍性贫血、血小板减少、巨幼贫、溶贫等)差异表达谱,差异表达结果用实时荧光定量PCR验证.共建立了5个基因的微球阵列,分别为Rap1GAP、RAC2、SPA1、RhoBTB3和内参GAPDH,每个基因检测的线性范围为0.002 5～0.1μmol,液相表达阵列具有良好的特异性和重复性(P<0.001).检测RA、RAEB、RAEBt、AML和其他组差异表达发现,RAC2、RhoBTB3、SPA-1和Rap1GAP各组间有显著性差异性存在(分别为P<0.000 1,P=0.049 1,P=0.020 6和P=0.004 6),其差异显著性与实时荧光定量PCR一致,泊松相关系数分别为0.930,0.946,0.945和0.921,具显著性(P<0.001).结果表明,成功建立了基于荧光微球的液相基因表达阵列,其敏感性高、特异性强、重复性好.     

High-fidelity mRNA amplification for gene profiling

The completion of the Human Genome Project has made possible the comprehensive analysis of gene expression, and cDNA microarrays are now being employed for expression analysis in cancer cell lines or excised surgical specimens. However, broader application of cDNA microarrays is limited by the amount of RNA required: 50-200 microg of total RNA (T-RNA) and 2-5 microg poly(A) RNA. To broaden the use of cDNA microarrays, some methods aiming at intensifying fluorescence signal have resulted in modest improvement. Methods devoted to amplifying starting poly(A) RNA or cDNA show promise, in that detection can be increased by orders of magnitude. However, despite the common use of these amplification procedures, no systematic assessment of their limits and biases has been documented. We devised a procedure that optimizes amplification of low-abundance RNA samples by combining antisense RNA (aRNA) amplification with a template-switching effect (Clonetech, Palo Alto, CA). The fidelity of aRNA amplified from 1:10,000 to 1:100,000 of commonly used input RNA was comparable to expression profiles observed with conventional poly(A) RNA- or T-RNA-based arrays.     

DNA array profiling of gene expression changes during maize embryo development

We are using DNA microarray-based gene expression profiling to classify temporal patterns of gene expression during the development of maize embryos, to understand mRNA-level control of embryogenesis and to dissect metabolic pathways and their interactions in the maize embryo. Genes involved in carbohydrate, fatty acid, and amino acid metabolism, the tricarboxylic acid (TCA) cycle, glycolysis, the pentose phosphate pathway, embryogenesis, membrane transport, signal transduction, cofactor biosynthesis, photosynthesis, oxidative phosphorylation and electron transfer, as well as 600 random complementary DNA (cDNA) clones from maize embryos, were arrayed on glass slides. DNA arrays were hybridized with fluorescent dye-labeled cDNA probes synthesized from kernel and embryo poly(A)⁺RNA from different stages of maize seed development. Several characteristic developmental patterns of expression were identified and correlated with gene function. Patterns of coordinated gene expression in the TCA cycle and glycolysis were analyzed in detail. The steady state level of poly(A)⁺ RNA for many genes varies dramatically during maize embryo development. Expression patterns of genes coding for enzymes of fatty acid biosynthesis and glycolysis are coordinately regulated during development. Genes of unknown function may by assigned a hypothetical role based on their patterns of expression resembling well characterized genes. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s10142-002-0046-6. Electronic Publication     

Multiplex degenerate PCR coupled with an oligo sorbent array for human endogenous retrovirus expression profiling

Human endogenous retroviruses (HERVs) can be divided into distinct families of tens to thousands of paralogous loci. The expression of HERV elements has been detected in all tissues tested to date, particularly germ cells, embryonic tissues and neoplastic tissues. Hence, the study of HERV expression could represent added value in cancer diagnosis. We developed a quantitative assay combining a multiplex degenerate PCR (MD-PCR) amplification, based on the relative conservation of the pol genes, and a colorimetric Oligo Sorbent Array (OLISA^®). Nine HERV families were selected and amplification primers and capture probes were designed for each family. The features required to achieve efficient amplification of most of the elements of each HERV family and balanced co-amplification of all HERV families were analyzed. We found that MD-PCR reliability, i.e. equivalence of amplification and dose-effect relationship, relied on the adjustment of three critical parameters: the primer degeneracy, the relative concentration of each primer and the total amount of primers in the amplification mixture. The analysis of tumoral versus normal tissues suggests that this assay could prove useful in tumor phenotyping.     

A quality-controlled microarray method for gene expression profiling

Gene expression profiling on microarrays is widely used to measure the expression of large numbers of genes in a single experiment. Because of the high cost of this method, feasible numbers of replicates are limited, thus impairing the power of statistical analysis. As a step toward reducing technically induced variation, we developed a procedure of sample preparation and analysis that minimizes the number of sample manipulation steps, introduces quality control before array hybridization, and allows recovery of the prepared mRNA for independent validation of results. Sample preparation is based on mRNA separation using oligo(dT) magnetic beads, which are subsequently used for first-strand cDNA synthesis on the beads. cDNA covalently bound to the magnetic beads is used as template for second-strand cDNA synthesis, leaving the intact mRNA in solution for further analysis. The quality of the synthesized cDNA can be assessed by quantitative polymerase chain reaction using 3'- and 5'-specific primer pairs for housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase. Second-strand cDNA is chemically labeled with fluorescent dyes to avoid dye bias in enzymatic labeling reactions. After hybridization of two differently labeled samples to microarray slides, arrays are scanned and images analyzed automatically with high reproducibility. Quantile-normalized data from five biological replica display a coefficient of variation 45% for 90% of profiled genes, allowing detection of twofold changes with false positive and false negative rates of 10% each. We demonstrate successful application of the procedure for expression profiling in plant leaf tissue. However, the method could be easily adapted for samples from animal including human or from microbial origin.     

Gene Expression Commons: an open platform for absolute gene expression profiling

Gene expression profiling using microarrays has been limited to comparisons of gene expression between small numbers of samples within individual experiments. However, the unknown and variable sensitivities of each probeset have rendered the absolute expression of any given gene nearly impossible to estimate. We have overcome this limitation by using a very large number (>10,000) of varied microarray data as a common reference, so that statistical attributes of each probeset, such as the dynamic range and threshold between low and high expression, can be reliably discovered through meta-analysis. This strategy is implemented in a web-based platform named "Gene Expression Commons" (https://gexc.stanford.edu/) which contains data of 39 distinct highly purified mouse hematopoietic stem/progenitor/differentiated cell populations covering almost the entire hematopoietic system. Since the Gene Expression Commons is designed as an open platform, investigators can explore the expression level of any gene, search by expression patterns of interest, submit their own microarray data, and design their own working models representing biological relationship among samples.     

Sharing gene expression data: an array of options

Sharing of microarray data has many advantages for the scientific and biomedical community, and should be advocated by neuroscience journals. The goals of sharing are manifold, and include improving analysis and confidence in results, and facilitating global comparisons between experiments, while at the same time, not penalizing those who share. The sharing of microarray data poses unique challenges relative to more generic data such as DNA sequences. These challenges are surmountable, and various sharing formats are possible. Centralized non-commercial databases are being developed to facilitate this process.     

Lack of association between an ecNOS gene polymorphism and diabetic nephropathy in type 2 diabetic patients with proliferative diabetic retinopathy.

The development of diabetic nephropathy shows remarkable variation among individuals. Therefore, not only hyperglycemia but also genetic factors may contribute to the development of diabetic nephropathy. The aim of the present study was to examine the contribution of the 27-bp repeat polymorphism in intron 4 of the endothelial constitutive nitric oxide synthase gene (ecNOS4) to the development of diabetic nephropathy. For this purpose, we analyzed this polymorphism in 167 Japanese type 2 diabetic patients with proliferative diabetic retinopathy consisting of 102 patients with diabetic nephropathy (with macroalbuminuria) and 65 patients without diabetic nephropathy (with normoalbuminuria). The genotype and allele frequencies were not significantly different between patients with diabetic nephropathy and those without diabetic nephropathy (ecNOS4 "b/b" 79.4% vs. 84.6%, ecNOS4 "b/a" 20.6% vs. 15.4%, "b" allele 89.7% vs. 92.3%, "a" allele 10.3% vs. 7.7%). We conclude that the ecNOS4 polymorphism does not contribute to the development of diabetic nephropathy.     

MedMiner: an Internet text-mining tool for biomedical information, with application to gene expression profiling

The trend toward high-throughput techniques in molecular biology and the explosion of online scientific data threaten to overwhelm the ability of researchers to take full advantage of available information. This problem is particularly severe in the rapidly expanding area of gene expression experiments, for example, those carried out with cDNA microarrays or oligonucleotide chips. We present an Internet-based hypertext program, MedMiner, which filters and organizes large amounts of textual and structured information returned from public search engines like GeneCards and PubMed. We demonstrate the value of the approach for the analysis of gene expression data, but MedMiner can also be extended to other areas involving molecular genetic or pharmacological information. More generally still, MedMiner can be used to organize the information returned from any arbitrary PubMed search.     

VizStruct: exploratory visualization for gene expression profiling

MOTIVATION: DNA arrays provide a broad snapshot of the state of the cell by measuring the expression levels of thousands of genes simultaneously. Visualization techniques can enable the exploration and detection of patterns and relationships in a complex data set by presenting the data in a graphical format in which the key characteristics become more apparent. The dimensionality and size of array data sets however present significant challenges to visualization. The purpose of this study is to present an interactive approach for visualizing variations in gene expression profiles and to assess its usefulness for classifying samples. RESULTS: The first Fourier harmonic projection was used to map multi-dimensional gene expression data to two dimensions in an implementation called VizStruct. The visualization method was tested using the differentially expressed genes identified in eight separate gene expression data sets. The samples were classified using the oblique decision tree (OC1) algorithm to provide a procedure for visualization-driven classification. The classifiers were evaluated by the holdout and the cross-validation techniques. The proposed method was found to achieve high accuracy. AVAILABILITY: Detailed mathematical derivation of all mapping properties as well as figures in color can be found as supplementary on the web page http://www.cse.buffalo.edu/DBGROUP/bioinformatics/supplementary/vizstruct. All programs were written in Java and Matlab and software code is available by request from the first author.