Experimental genomics:The application of DNA microarrays in cellular and molecular biology studies

The genome sequence information in combination with DNA microarrays promises to revolutionize the way of cellu-lar and molecular biological research by allowing complex mixtures of RNA and DNA to interrogated in a parallel and quantita-tive fashion. DNA microarrays can be used to measure levels of gene expression for tens of thousands of gene simultane-ously and take advantage of all available sequence information for experimental design and data interpretation in pursuit of biological understanding. Recent progress in experimental genomics allows DNA microarrays not simply to provide a cata-logue of all the genes and information about their function, but to understand how the components work together to comprise functioning cells and organisms. This brief review gives a survey of DNA microarrays technology and its applications in ge-nome and gene function analysis, gene expression studies, biological signal and defense system, cell cycle regulation, mechanism of transcriptional regulation, proteomics, and the functionality of food component.

Prospects of DNA microarray application in management of chronic obstructive pulmonary disease:A systematic review

Chronic obstructive pulmonary disease(COPD)is incurable chronic disease which kills 3.3 million each year worldwide.Number of global cases of COPD is steadily rising alongside with life expectancy,disproportionally hitting middle-income countries like Russia and China,in such conditions,new approaches to the COPD management are desperately needed.DNA microarray technology is a powerful genomic tool that has the potential to uncover underlying COPD biological alteration and brings up revolutionized treatment option to clinicians.We executed systematic review studies of studies published in last 10 years regarding DNA microarray application in COPD management,with complacence to PRISMA criteria and using PubMed and Medline data bases as data source.Out of 920 identified papers,39 were included in the final analysis.We concluded that Genome-wide expression profiling using DNA microarray technology has great potential in enhancing COPD management.Current studied proofed this method is reliable and possesses many potential applications such as individual at risk of COPD development recognition,early diagnosis of disease,COPD phenotype identification,exacerbation prediction,personalized treatment optioning and prospect of oncogenesis evaluation in patients with COPD.Despite all the proofed benefits of this technology,researchers are still in the early stage of exploring it’s potential.Therefore,large clinical trials are still needed to set up standard for DNA microarray techniques usage implementation in COPD management guidelines,subsequently giving opportunity to clinicians for controlling or even eliminating COPD entirely.

Detection of Genetically Modified Crops by Combination of Multiplex PCR and Low-density DNA Microarray

Objective To develop a technique for simultaneous detection of various target genes in Roundup Ready soybean by combining multiplex PCR and low-density DNA microarray. Methods Two sets of the multiplex PCR system were used to amplify the target genes in genetically modified （GM） soybean. Seventeen capture probes （PCR products） and 17 pairs of corresponding primers were designed according to the genetic characteristics of Rroundup Ready soybean （GTS40-3-2）, maize （MonS10, Nk603, GA21）, canola （T45, MS1/RF1）, and rice （SCK） in many identified GM crops. All of the probes were categorized and identified as species-specific probes. One negative probe and one positive control probe were used to assess the efficiency of all reactions, and therefore eliminate any false positive and negative results. After multiplex PCR reaction, amplicons were adulterated with Cy5-dUTP and hybridized with DNA microarray. The array was then scanned to display the specific hybridization signals of target genes. The assay was applied to the analysis of sample of certified transgenic soybean （Roundup Ready GTS40-3-2） and canola （MS1/RF1）. Results A combination technique of multiplex PCR and DNA microarray was successfully developed to identify multi-target genes in Roundup Ready soybean and MS 1/RF1 canola with a great specificity and reliability. Reliable identification of genetic characteristics of Roundup Ready of GM soybean from genetically modified crops was achieved at 0.5% transgenic events, indicating a high sensitivity. Conclusion A combination technique of multiplex PCR and low-density DNA microarray can reliably detect and identify the genetically modified crops.

Subtype Identification of Avian Influenza Virus on DNA Microarray

We have developed a rapid microarray-based assay for the reliable detection of H5, H7 and H9 subtypes of avian influenza virus （AIV）. The strains used in the experiment were A/Goose/Guangdong/1/96 （H5N1）, A/African starling/983/79 （H7N1） and A/Turkey/Wiscosin/1/66 （H9N2）. The capture DNAs clones which encoding approximate 500-bp avian influenza virus gene fragments obtained by RT-PCR, were spotted on a slide-bound microarray. Cy5-labeled fluorescent cDNAs, which generated from virus RNA during reverse transcription were hybridized to these capture DNAs. These capture DNAs contained multiple fragments of the hemagglutinin and matrix protein genes of AIV respectively, for subtyping and typing AIV. The arrays were scanned to determine the probe binding sites. The hybridization pattern agreed approximately with the known grid location of each target. The results show that DNA microarray technology provides a useful diagnostic method for AIV.

Development and evaluation of a DNA microarray assay for the simultaneous detection of nine harmful algal species in ship ballast and seaport waters

Rapid,high-throughput and reliable methods are urgently required to accurately detect and monitor harmful algae,which are responsible for algal blooms,such as red and green tides. In this study,we successfully developed a multiplex PCR-based DNA microarray method capable of detecting nine harmful algal species simultaneously,namely A lexandrium tamarense,Gyrodinium instriatum,Heterosigma akashiwo,Karenia mikimotoi,Prorocentrum donghaiense,Prorocentrum minimum,Ulva compressa,Ulva ohnoi and Ulva prolifera. This method achieved a limit of detection(LOD) of 0.5 ng of genomic DNA(orders of magnitude of the deci-nanogram range) in the tested algae cultures. Altogether,230 field samples from ship ballast waters and seaport waters were used to evaluate the DNA microarray. The clinical sensitivity and specificity of the DNA microarray assay in detecting field samples were 96.4% and 90.9%,respectively,relative to conventional morphological methods. This indicated that this high-throughput,automatic,and specific method is well suited for the detection of algae in water samples.

Polyurethane Molecular Stamps for the in situ Synthesis of DNA Microarray

Fabrication of polyurethane molecular stamps (PU stamps) based on polypropylene glycol (PPG) and toluene diisocyanate (TDI), using 3, 3-dichloro-4, 4-methylenedianiline (MOCA) as the crosslinker, is reported. It was shown from the contact angle measurement that PU stamps surface has good affinity with acetonitrile, guaranteeing the well distribution of DNA monomers on patterned stamps. Laser confocal fluorescence microscopy images of oligonucleotide arrays after hybridization confirmed polyurethane is an excellent material for molecular stamps when transferring polar chemicals and conducting reactions on interfaces by stamping.

Endonuclease-based Method for Detecting the Sequence Specific DNA Binding Protein on Double-stranded DNA Microarray

The double-stranded DNA (dsDNA) probe contains two different protein binding sites. One is for DNA- binding proteins to be detected and the other is for a DNA restriction enzyme. The two sites were arranged together with no base interval. The working principle of the capturing dsDNA probe is described as follows: the capturing probe can be cut with the DNA restriction enzyme (such as EcoR I) to cause a sticky terminal, if the probe is not bound with a target protein, and the sticky terminal can be extended and labeled with Cy3-dUTP by DNA polymerase. When the probe is bound with a target protein, the probe is not capable to be cut by the restriction enzyme because of space obstruction. The amount of the target DNA binding proteins can be measured according to the variations of fluorescent signals of the corresponding probes.

PROGRESS IN DNA CHIP TECHNOLOGY

DNA chip technology employs light- directed in situ oligonucleotide synthesis and/or DNA microarray printing device to produce arrays of large number of probes in the tiny surface of silicon substrates, which makes it possible that the gene detection be conducted efficiently with high speed and sensitivity. The DNA chip may take important part in genome research, gene diagnoses and so on.

Aquaporin-8 expression is reduced in ileum and induced in colon of patients with ulcerative colitis

AIM： To study susceptibility genes which may play a potential role in the pathogenesis and etiology of inflammatory bowel disease （IBD）. METHODS： To identify potential susceptibility genes we performed global gene expression profiling in patients with IBD and control specimens. For determination of an intrinsic gene expression profile in ulcerative colitis （UC） and Crohn＇s disease （CD） compared to normal subjects, mucosal biopsies of non-inflamed regions of the colon and the terminal ileum were subjected to DNA microarray analysis. Real-time RT-PCR and immunohistochemistry were used for verification of selected regulated candidate genes and a genetic analysis was performed. RESULTS： We could show that aquaporin-8 （AQP8） mRNA and protein levels were significantly increased in the colon of UC patients compared to controls. Genetic analysis of the six exons and the promoter region of AQPS, however, revealed no mutations or polymorphisms in IBD patients. CONCLUSION： Our results suggest that upregulation of AQP8 in the colon of UC patients represents a secondary phenomenon which may, due to altered water exchange of the distal intestinal mucosa, disturb the physiologic colonic mucus barrier and thus lead to chronic inflao mmation and ulceration.

Expression of a novel bHLH-Zip gene in human testis

<abstract>Aim: To identify specifically expressed genes in the adult and fetal testes. Methods: A human testis cDNA microarray was established. Then the mRNA of adult and fetal testis was purified and probes were prepared by a reverse transcription reaction with the testis mRNA as template. The microarray was hybridized with probes of adult and fetal testes. The nucleic sequences of differentially expressed genes were determined and homologies were searched in the databases of the GenBank. Results: When hybridized with adult or fetal testis probes, the positive clones were 96.8 % and 95.4 %, respectively. Among these genes, one was a new testis-specific gene, which was named TSP1. TSP1 was highly expressed in human adult testis. The cDNA of TSP1 was 1,484 bp in length. The cDNA sequence of this clone was deposited in the Genbank (AF333098). TSP1 was also determined as Interim Gen Symbol (Unigene, No. Hs.98266). Protein analysis showed that TSP1 contained two functional domains: an N-terminal basic helix-loop-helix (bHLH) and a C-terminal leucine zipper (Zip). Homologous analysis showed that the 430 amino acid sequences deduced from the 1,293 bp open reading frame (ORF) had a homology with the human gene FLJ2509 (AK098575). TSP1 had also a sequence homology with Spz 1 protein of mouse. Expression profiles showed that TSP1 was specifically and strongly expressed in the testis. Conclusion: TSP1 is a gene highly expressed in adult testis. It may play an important role in spermatogenesis in the humans.

Cancer/testis antigens： novel tools for discerning aggressive and non-aggressive prostate cancer

The introduction of serum prostate-specific antigen （PSA） in the 1980s has dramatically altered and benefited the initial diagnosis of prostate cancer. However, the widespread use of PSA testing has resulted in overdetection and overtreatment of potentially indolent disease. Thus, a clinical dilemma today in the management of prostate cancer is to discern men with aggressive disease who need definitive treatment from men whose disease are not lethal. Although several serum and tissue biomarkers have been evaluated during the past decade, improved markers are still needed to enhance the accuracy, with which patients at risk can be discerned and treated more aggressively. The cancer/testis antigens （CTAs） are a group of proteins that are restricted to the testis in the normal adult, but are aberrantly expressed in several types of cancers. Because of their restricted expression pattern, the CTAs represent attractive biomarker candidates for cancer diagnosis/prognosis. Furthermore, several studies to date have reported the differential expression of CTAs in prostate cancer. Here, we review recent developments that demonstrate the potential of the CTAs as biomarkers to discern the a^ressive Dhenotvoe of orostate cancer.

Effects of Aging and Advanced Glycation on Gene Expression in Cerebrum and Spleen of Mice

Objective To analyze the effects of aging or advanced glycation on gene expression in the cerebrum and spleen of female C57BL/6J mice. Methods The gene expression profile was determined by using cDNA expression arrays containing 588 cDNA. Results Aging and advanced glycation resulted in differential gene expression patterns of cerebrum and spleen compared with young mice. Among the 80 genes detected in cerebrum, 43 exhibited a change in mRNA ratios with aging or treatment. Thirty-four changes (79%) were common in aged and D-galactose treated mice, whereas the cerebrum from aged and AGE-lysine treated mice showed common changes in expression of 38 genes (88%). Of the 86 genes detected in spleen, 29 (34%) displayed an age-related decrease in expression, whereas 3 (3%) displayed an increase in expression levels with aging. Eighteen genes from the detectable genes exhibited expression changes in both cerebrum and spleen of mice. Conclusions The gene expression profiles of D-galactose and AGE-lysine treated mice resemble those of aged mice. Use of cDNA hybridization arrays may provide a promising tool to explore the mechanism of aging at a molecular level.

A novel strategy for in situ maskless synthesis of biochips:Self-driving micro-fluid porous type printing (SMPTP)

A novel maskless technique, self-driving micro-fluid porous type printing （SMPTP）, was reported to in situ synthesize oligonucleotide arrays on glass slide, which has the merits of low cost, high quality and simple craft. In SMPTP for fabricating gene- chips, porous fiber tubes with a number of nanometric or micron channels functioned as ＂active letters＂ and were assembled in designed patterns, which are identical to the distribution of monomers in each layer of the array, and four patterns were needed for each layer. By means of capillarity, the synthesis solution was automatically taken into porous tubes assembled in a printing plate and reached the surface. An oligonucleotide array of 160 features with four different 15-mer probes was in situ synthesized using this technique. The four specific oligonucleotide probes, including the matched and the mismatched by the fluorescent target sequence, gave obviously different hybridization fluorescent signals.

Expressed genes in regenerating rat liver after partial hepatectomy

AIM: To reveal the liver regeneration (LR) and its controlas well as the occurrence of liver disease and to study the gene expression profiles of 551 genes after partial hepatectomy (PH) in regenerating rat livers.METHODS: Five hundred and fifty-one expressed sequence tags screened by suppression subtractive hybridization were made into an in-house cDNA microarray, and the expressive genes and their expressive profiles in regenerating rat livers were analyzed by microarray and bioinformatics. RESULTS: Three hundred of the analyzed 551 genes were up- or downregulated more than twofolds at one or more time points during LR. Most of the genes were up- or downregulated 2-5 folds, but the highest reached 90 folds of the control. One hundred and thirty-nine of themshowed upregulation, 135 displayed downregulation, and up or down expression of 26 genes revealed a dependence on regenerating livers. The genes expressedin 24-h regenerating livers were much more than those in the others. Cluster analysis and generalization analysis showed that there were at least six distinct temporal patterns of gene expression in the regenerating livers, that is, genes were expressed in the immediate early phase, early phase, intermediate phase, early-late phase, late phase, terminal phase. CONCLUSION: In LR, the number of down-regulated genes was almost similar to that of the upregulated genes; the successively altered genes were more than the rapidly transient genes. The temporal patterns of gene expression were similar 2 and 4 h, 12 and 16 h, 48 and 96 h, 72 and 144 h after PH. Microarray combined with suppressive subtractive hybridization can effectively identify the genes related to LR.

Effects of different ingredients of zedoary on gene expression of HSC-T6 cells

AIM： To investigate the effects of four different ingredients of zedoary （Curcuma aromatica oil, Curcumol, β-elemence, and Curcumin） on the gene expressions of hepatic stellate cells （HSCs）, and to explore the molecular mechanism of zedoary against hepatic fibrosis at gene network level. METHODS： We detected the mRNA sequences of 50 liver fibrosis-related genes in GenBank and designed oligonucleotide probes. We synthesized oligonucleotides with PE8909 DNA synthesizing instrument, and carried out oligonucleotide microarray with OGR-04 dropping instrument and aldehyded glass chip. Cultured HSC-T6 cells were breated wibh different concentrations of Colchicine, Curcuma aromatica oil, Curcumol, β-elemence, and Curcumin. According to the experiment of cell toxicity, we took the appropriate concentrations of medicines that resulted in over 50% of cell survival as experiment concentrations. We collected the cells at 1, 6, 12, and 24 h, and extracted total RNA with TRIzol reagent, then labeled cDNAs with Cy3-dUTP and Cy5-dUTP. These labeled cDNAs were hybridized to an oligonucleotide microarray which was washed several times and scanned by scanner GenePix 4000B. Different gene expressions of HSC-T6 cells were analyzed by ImaGene 4.2 software. RESULTS： After HSC-T6 cells were cultured in a medium containing 6.25μg/mL Colchicine for 12 h, expression of TIMP-1 decreased 2.2-folds. After HSC-T6 cells were cultured in a medium containing 78.125 μg/mL of Curcuma aromatica oil for 24 h, the expression of TIMP-2 and IL-6 decreased 2.3- and 2.2-folds, respectively. Moreover, after HSC-T6 cells were cultured in a medium containing 1.5625 μg/mL of Curcumol for 12 h, the expression of TGFμ1 and P450a decreased 2.3- and 2.1-folds, respectively. CONCLUSION： Our results may show the possible molecular mechanism of Curcuma aromatica oil and Curcumol against hepatic fibrosis.

RRAS:A key regulator and an important prognostic biomarker in biliary atresia

AIM:To characterize the differentially expressed gene profiles in livers from biliary atresia (BA) patients including,ascertain genes,functional categories and pathways that play a central role in the pathogenesis of BA,and identify the novel prognostic markers for BA.METHODS:Liver tissue samples from control patients,neonatal cholestasis patients,and BA patients at the age of < 60 d,60-90 d,and > 90 d were pooled for DNA microarray analysis.Bioinformatics analysis was performed using,series test cluster of gene ontology,and Pathway-Finder software.Reverse-transcription polymerase chain reaction was performed to confirm changes in selected genes.Relation between RRAS gene expression and prognosis of 40 BA patients was analyzed in a 2-year follow-up study.RESULTS:The 4 identified significant gene expression profiles could confidently separate BA liver tissue from normal and other diseased liver tissues.The included genes were mainly involved in inflammation response and reconstruction of cellular matrix.The significant pathways associated with BA were primarily involved in autoimmune response,activation of T lymphocytes and its related cytokines.The RRAS,POMC,SLC26A6 and STX3 genes were important regulatory modules in pathogenesis of BA.The expression of RRAS was negatively correlated with the elimination rate of jaundice and positively correlated with the occurrence rate of cholangitis.CONCLUSION:Autoimmune response mediated by T lymphocytes may play a vital role in the pathogenesis of BA.The RRAS gene is an important regulatory module in the pathogenesis of BA,which may serve as a novel prognostic marker for BA.

Analysis of Gene Expression Pattern of Lumbar Intervertebral Disc Degeneration in Human

ObjectiveTo investigate the gene expression changes in normal and degeneration lumbar intervertebral disc in humans, providing information for clinical. MethodsThe PCR products of 4096 human genes were spotted onto a kind of chemical-material-coated-glass slides. The total RNAs were isolated from the tissues. Both the mRNAs from the degeneration and normal lumbar intervertebral disc in humans were reversely transcribed to the cDNAs, which used as the hybridization probes with the incorporations of fluorescent dUTP. The mixed probes were then hybridized to the cDNA microarray. After high-stringent washing, the cDNA microarray was scanned for the fluorescent signals and analyzed with computer image analysis. ResultsAmong the 4096 targets, there were 706 genes whose expression levels differed between the degeneration and normal lumbar intervertebral disc in all cases, comprising 298 up-regulated and 358 down-regulated ones. ConclusionDNA microarray technology is an effective technique in screening for differently expressed genes between the degeneration and normal lumbar intervertebral disc. Cell apoptosis plays an important role in the process of lumbar intervertebral disc degeneration.

A New Optimized Wrapper Gene Selection Method for Breast Cancer Prediction

Machine-learning algorithms have been widely used in breast cancer diagnosis to help pathologists and physicians in the decision-making process.However,the high dimensionality of genetic data makes the classification process a challenging task.In this paper,we propose a new optimized wrapper gene selection method that is based on a nature-inspired algorithm(simulated annealing(SA)),which will help select the most informative genes for breast cancer prediction.These optimal genes will then be used to train the classifier to improve its accuracy and efficiency.Three supervised machine-learning algorithms,namely,the support vector machine,the decision tree,and the random forest were used to create the classifier models that will help to predict breast cancer.Two different experiments were conducted using three datasets:Gene expression(GE),deoxyribonucleic acid(DNA)methylation,and a combination of the two.Six measures were used to evaluate the performance of the proposed algorithm,which include the following:Accuracy,precision,recall,specificity,area under the curve(AUC),and execution time.The effectiveness of the proposed classifiers was evaluated through comprehensive experiments.The results demonstrated that our approach outperformed the conventional classifiers as expected in terms of accuracy and execution time.High accuracy values of 99.77%,99.45%,and 99.45%have been achieved by SA-SVM for GE,DNA methylation,and the combined datasets,respectively.The execution time of the proposed approach was significantly reduced,in comparison to that of the traditional classifiers and the best execution time has been reached by SA-SVM,which was 0.02,0.03,and 0.02 on GE,DNA methylation,and the combined datasets respectively.In regard to precision and specificity,SA-RF obtained the best result of 100 on GE dataset.While SA-SVM attained the best recall result of 100 on GE dataset.

A specific gene-expression signature quantifies the degree of hepatic fibrosis in patients with chronic liver disease

AIM： To study a more accurate quantification of hepatic fibrosis which would provide dinically useful information for monitoring the progression of chronic liver disease. METHODS： Using a cDNA microarray containing over 22000 clones, we analyzed the gene-expression profiles of non-cancerous liver in 74 patients who underwent hepatic resection. We calculated the ratio of azanstained： total area, and determined the morphologic fibrosis index （MFI）, as a mean of 9 section-images. We used the MFI as a reference standard to evaluate our method for assessing liver fibrosis. RESULTS： We identified 39 genes that collectively showed a good correlation （r 〉 0.50） between geneexpression and the severity of liver fibrosis. Many of the identified genes were involved in immune responses and cell signaling. To quantify the extent of liver fibrosis, we developed a new genetic fibrosis index （GFI） based on gene-expression profiling of 4 clones using a linear support vector regression analysis. This technique, based on a supervised learning analysis, correctly quantified the various degrees of fibrosis in both 74 training samples （r = 0.76, 2.2% vs 2.8%, P 〈 0.0001） and 12 independent additional test samples （r = 0.75, 9.8% vs 8.6%, P 〈 0.005）. It was far better in assessing liver fibrosis than blood markers such as prothrombin time （r = -0.53）, type IV collagen 7s （r = 0.48）, hyaluronic acid （r = 0.41）, and aspartate aminotransferase to platelets ratio index （APRI） （r = 0.38）. CONCLUSION： Our cDNA microarray-based strategy may help clinicians to precisely and objectively monitor the severity of liver fibrosis.

Cluster Analysis of Differentially Expressed Heat Stress Genes in Rat Jejunal Mucosal

[Objective] The aim was to study the heat stress mechanism of differen tially expressed genes in rat jejunal mucosal.[Method] Variable cluster analysis and cluster analysis of samples on the differentially expressed heat stress genes in rat jejunal mucosal were carried out with SAS software,and statistics of distribution of the differentially expressed genes on chromosomes were conducted.[Result] The differentially expressed genes were divided into seven categories,of which,the upregulated genes included three categories （i.e.：Category A：Hspa1a,Hspa1b,Hspb1,Hsph1,Dnaja4,Ahsa2 and P4ha1; Category B：Cyp1a2,Zbtb16,Gucy2g,Fgb,Cyp4a3 and Etv2; and Category C：Cyp1a2,Chac1 and Cyp4b1） and the down-regulated genes included four categories （i.e.：Category D：Tlr2,Noxo1,LOC286989 and Aspg; Category E：RGD1560395,Alb and BQ194726; Category F：Ccl4,Gzmk,Al228153,Anxa10,S100a9 and Ascl5; and Category G：Reg1a and Slc13a1）.The classification,function and reasons of differential expression for each gene category were analyzed.[Conclusion] Most of the three categories of up-regulated genes were related to the heat shock proteins; and most of the four categories of down-regulated genes were related to the immunity,providing reference for discussion of the heat stress mechanism.