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PubMed2005

A robust statistical method for detecting differentially expressed genes.

DNA microarray technology allows researchers to monitor the expressions of thousands of genes under different conditions, and to measure the levels of thousands of different DNA molecules at a given point in the life of an organism, tissue or cell. A wide variety of different diseases that are characterised by unregulated gene expression, DNA replication, cell division and cell death, can be detected early using microarrays. One of the major objectives of microarray experiments is to identify differentially expressed genes under various conditions. The detection of differential gene expression under two different conditions is very important in biological studies, and allows us to identify experimental variables that affect different biological processes. Most of the tests available in the literature are based on the assumption of normal distribution. However, the assumption of normality may not be true in real-life data, particularly with respect to microarray data.A test is proposed for the identification of differentially expressed genes in replicated microarray experiments conducted under two different conditions. The proposed test does not assume the distribution of the parent population; thus, the proposed test is strictly nonparametric in nature. We calculate the p-value and the asymptotic power function of the proposed test statistic. The proposed test statistic is compared with some of its competitors under normal, gamma and exponential population setup using the Monte Carlo simulation technique. The application of the proposed test statistic is presented using microarray data. The proposed test is robust and highly efficient when populations are non-normal.

Journal Article↗

PubMed2002

[The use of gene expression differential display analysis technics in trauma research].

Gene is the regulation core of cell proliferation, differentiation and maturation. It is also the decisive factor of occurrence, progression and prognosis of many diseases. The changes in gene expression will inevitably lead to many kinds of abnormal in cells, tissue, organs even in whole biological organism. Trauma, as well as other stimulators, may bring a series complications through the abnormal expression of genes following injury. With the development of bioinformatics and molecular biology, a series of assays (so called gene expression differential display analysis technics, such as DNA microarray) have been established, which have been used to analyze differentially expressed genes effectively. These methods have been widely used in the research of tumor and other disease. This review will focus to some usage of the ways in traumatology.

Animals↗

PubMed2001

Improvement of RNA fingerprinting efficiency for the analysis of differential gene expression in human cardiac macro- and microvascular endothelial cells.

RNA fingerprinting by arbitrarily primed PCR (RAP-PCR) is a powerful tool to screen differential gene expression. However, PCR-based screening techniques show a high incidence of false positive results (40-90%). In order to increase the efficiency and feasibility of RAP-PCR, the original protocol was modified and applied to analyse differential gene expression in human coronary macro- (HCEC) and microvascular (HCMEC) endothelial cells. The major modifications introduced were: (i) the use of two primers for PCR amplification, instead of reverse-transcription primer alone; (ii) the use of three cycles at low stringency followed by further amplification at high stringency; (iii) optimization of amplification cycle number, template amount, and concentration of primers, dNTP, Mg(2+); (iv) detection of fingerprints by silver staining; and (v) direct sequencing using RAP-PCR primers. Analysis of untreated and TNF alpha -stimulated (100 U ml(-1)for 1, 4, and 24 h) HCEC and HCMEC displayed 11 differentially expressed products by 18 primer combinations. Confirmation of results by RT-PCR showed that the rate of false positives attributable to our screening method was less than 20%. Among detected RAP-PCR products, the expression of Mn-superoxide dismutase, A20 zinc finger protein, and three novel genes (A/a, 4/d, 7/c) was more strongly modulated by TNF in HCEC than HCMEC. A further novel gene (B/e) was strongly expressed in HCMEC while only barely detectable in HCEC. In conclusion, modification of RAP-PCR strongly reduced the incidence of false positives, eliminated a radioactive requirement, and allowed sequencing without prior cloning, supplying an improved technology able to identify new differentially expressed genes between macro- and microvascular endothelial cells.

Cells, Cultured↗

PubMed1998

Sp1 as a target site for metal-induced perturbations of transcriptional regulation of developmental brain gene expression.

Differential gene expression is partially regulated by zinc finger proteins (ZFP) such as Sp1, which may be potential targets for perturbations by environmental metals. In this paper, we discuss the selective effects of lead (Pb) and other heavy metals on the in vitro and in vivo DNA-binding of Sp1, and the developmental expression of its target genes. We have found that the presence of Pb, Zn and Cd in a DNA-binding assay differentially modulated the binding of Sp1 to its specific DNA sequence, while Ca, Mg and Ba, did not. In PC12 cells, cultured in the presence of low concentrations of Pb, a premature enhancement of Sp1 DNA-binding was observed. Similarly, Sp1 DNA-binding in the cerebellum of Pb-exposed animals was shifted to the first week after birth, while the developmental profile of a non-ZFP, NFkB, was not. Furthermore, selective premature peaks of myelin basic protein and proteolipid protein mRNA expression were observed to occur in a manner relative to the changes in Sp1 DNA-binding. Since these genes are high targets for Sp1, these data suggest that exposure to heavy metals may alter developmental gene expression and brain development through selective modulation of the transcriptional activity of Sp1.

Animals↗

PubMed2005

Tuber on a chip: differential gene expression during potato tuber development.

Potato tuber development has proven to be a valuable model system for studying underground sink organ formation. Research on this topic has led to the identification of many genes involved in this complex process and has aided in the unravelling of the mechanisms underlying starch synthesis. However, less attention has been paid to the biochemical pathways of other important metabolites or to the changing metabolic fluxes occurring during potato tuber development. In this paper, we describe the construction of a potato complementary DNA (cDNA) microarray specifically designed for genes involved in processes related to tuber development and tuber quality traits. We present expression profiles of 1315 cDNAs during tuber development where the predominant profiles were strong up- and down-regulation. Gene expression profiles showing transient increases or decreases were less abundantly represented and followed more moderate changes, mainly during tuber initiation. In addition to the confirmation of gene expression patterns during tuber development, many novel differentially expressed genes were identified and are considered as candidate genes for direct involvement in potato tuber development. A detailed analysis of starch metabolism genes provided a unique overview of expression changes during tuber development. Characteristic expression profiles were often clearly different between gene family members. A link between differential gene expression during tuber development and potato tissue specificity is described. This dataset provides a firm basis for the identification of key regulatory genes in a number of metabolic pathways that may provide researchers with new tools to achieve breeding goals for use in industrial applications.

Journal Article↗

PubMed2002

Differential gene expression in vascular smooth muscle cells in primary atherosclerosis and in stent stenosis in humans.

OBJECTIVE: We sought to identify differentially expressed genes in human in stent stenosis (ISS) to provide insights into the mechanism of disease. METHODS AND RESULTS: Using representation difference analysis, we examined differential gene expression between cultured normal human medial vascular smooth muscle cells (VSMCs) and cells from primary atherosclerotic plaques or ISS sites. Specific groups of genes were overexpressed in ISS and plaque VSMCs, including cell cycle regulatory proteins and cell matrix and contractile proteins. Differential expression was validated by virtual Northern analysis, reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunohistochemistry. All ISS genes were expressed by normal intima and had even higher expression in primary plaque VSMCs. CONCLUSIONS: ISS VSMCs have a stable gene expression profile reflecting an intimal pattern, intermediate between normal medial and primary plaque VSMCs. Differential expression profiling may identify markers of disease that are overexpressed in ISS and also help elucidate the origin of the ISS lesion.

Adult↗

PubMed2001

Identification of differentially expressed genes in human memory (CD45RO+) CD4+ T lymphocytes.

The precise nature and development of the memory (CD45RO+) CD4+ T lymphocytes remain unknown. In this study, we analyzed differential gene expression of human memory CD4+ T lymphocytes in relation to their naive counterparts. A suppression subtractive hybridization technique was used to isolate and clone differentially expressed genes in the memory subset with respect to the naive subset. We screened approximately 300 clones by dot blot analysis and sequenced 23 differentially expressed clones. GenBank sequence homology search showed that these clones included genes for transcription factors, enzymes and immunomodulatory molecules. Differential expression of a subset of these genes was further confirmed by RT-PCR and densitometric analysis revealed that they were expressed five to eightfold more in memory than naive CD4+ T lymphocytes. Collectively, these results suggest that multiple genes with different functions contribute to the development of immunological memory in human T lymphocytes.

CD4-Positive T-Lymphocytes↗

PubMed2004

Detection of chromosomal regions showing differential gene expression in human skeletal muscle and in alveolar rhabdomyosarcoma.

BACKGROUND: Rhabdomyosarcoma is a relatively common tumour of the soft tissue, probably due to regulatory disruption of growth and differentiation of skeletal muscle stem cells. Identification of genes differentially expressed in normal skeletal muscle and in rhabdomyosarcoma may help in understanding mechanisms of tumour development, in discovering diagnostic and prognostic markers and in identifying novel targets for drug therapy. RESULTS: A Perl-code web client was developed to automatically obtain genome map positions of large sets of genes. The software, based on automatic search on Human Genome Browser by sequence alignment, only requires availability of a single transcribed sequence for each gene. In this way, we obtained tissue-specific chromosomal maps of genes expressed in rhabdomyosarcoma or skeletal muscle. Subsequently, Perl software was developed to calculate gene density along chromosomes, by using a sliding window. Thirty-three chromosomal regions harbouring genes mostly expressed in rhabdomyosarcoma were identified. Similarly, 48 chromosomal regions were detected including genes possibly related to function of differentiated skeletal muscle, but silenced in rhabdomyosarcoma. CONCLUSION: In this study we developed a method and the associated software for the comparative analysis of genomic expression in tissues and we identified chromosomal segments showing differential gene expression in human skeletal muscle and in alveolar rhabdomyosarcoma, appearing as candidate regions for harbouring genes involved in origin of alveolar rhabdomyosarcoma representing possible targets for drug treatment and/or development of tumor markers.

Chromosome Mapping↗

PubMed2006

Systemic analysis of the differential gene expression profile in a colonic adenoma-normal SSH library.

BACKGROUND: The discovery of differentially expressed genes of colonic adenoma minus normal mucosa enables the understanding of early molecular events in colorectal carcinogenesis. In our previous study, we have developed an adenoma minus normal mucosa suppression subtractive hybridization (SSH) library and identified 109 differentially expressed clones. METHODS: An in-house EST pipeline and the Gene Ontology web-based tool () were used to analyze these clones. Realtime quantitative RT-PCR (Q-PCR) was applied to detect the expression of 14-3-3 zeta, REG4 and 6 ribosomal protein genes (RPS2, RPS12, RPS27A, RPL5, RPL7a and RPL10a) in 14 adenomas (8 with concurrent cancers) and 44 colorectal adenocarcinomas with paired normal mucosa. RESULTS: Sixty-two candidate genes were obtained from this library. Bioinformatics analysis indicated that both ribosomal protein genes and immune-related genes were enriched. REG4 was significantly upregulated in colorectal adenomas (medium fold: 1.676, p<0.05, Wilcoxon test) and 14-3-3 zeta in cancers (medium fold: 1.202, p<0.01, Wilcoxon test), as compared with those of paired normal mucosa. However, all ribosomal protein genes were not significantly overexpressed in colorectal adenomas or cancers. CONCLUSIONS: A differential gene expression profile in A-N SSH library may be helpful in understanding the molecular mechanism of colorectal cancer initiation and progression. REG4 and 14-3-3 zeta may be potential biomarkers for early colorectal cancer detection.

Adenoma↗

PubMed1996

The sea pansy Renilla reniformis luciferase serves as a sensitive bioluminescent reporter for differential gene expression in Candida albicans.

The infectious yeast Candida albicans progresses through two developmental programs which involve differential gene expression, the bud-hypha transition and high-frequency phenotypic switching. To understand how differentially expressed genes are regulated in this organism, the promoters of phase-specific genes must be functionally characterized, and a bioluminescent reporter system would facilitate such characterization. However, C. albicans has adopted a nontraditional codon strategy that involves a tRNA with a CAG anticodon to decode the codon CUG as serine rather than leucine. Since the luciferase gene of the sea pansy Renilla reinformis contains no CUGs, we have used it to develop a highly sensitive bioluminescent reporter system for C. albicans. When fused to the galactose-inducible promoter of GAL1, luciferase activity is inducible; when fused to the constitutive EF1 alpha 2 promoter, luciferase activity is constitutive; and when fused to the promoter of the white-phase-specific gene WH11 or the opaque-phase-specific gene OP4, luciferase activity is phase specific. The Renilla luciferase system can, therefore, be used as a bioluminescent reporter to analyze the strength and developmental regulation of C. albicans promoters.

Animals↗

PubMed1992

Differential gene expression between young and senescent, quiescent WI-38 cells.

To investigate age-related changes in gene expression in WI-38 cells, we isolated RNA from young and senescent, quiescent cultures and made subtracted cDNA libraries. Density-arrested cells were incubated in serum-free MCDB-104 for 3 days. RNA was then isolated and subtracted cDNA libraries were made in the phagemid vector pCDM8. Both by picking clones at random from these subtracted libraries and by differential hybridization screening with subtracted cDNA probes from young and senescent cells, we have identified a total of 11 genes for which RNA is expressed differentially in these quiescent young and senescent WI-38 cultures. Two genes, EPC-1 and EPC-A2, with elevated RNA levels in young cells, have sequences which have not previously been identified. Two of the genes with elevated RNA expression in the senescent cells are the mitochondria-coded genes for NADH dehydrogenase subunit 4 and for cytochrome b. We also identified seven other genes with elevated RNA levels in senescent cells. Three of these, LPC-1, LPC-14 and LPC-24, have been partially sequenced and have not previously been identified. These studies show that density-arrested, serum-deprived, quiescent young and senescent cells express a number of genes differentially. These differences are not growth-dependent, but are age-dependent. Our studies also show that the methods employed here, which include careful regulation of the cell cultures and subtraction of the libraries, result in libraries from which differentially expressed genes can be identified, either by random selection or by differential hybridization screening with subtracted probes.

Aging↗

PubMed2002

Differential gene expression in response to copper in Acidithiobacillus ferrooxidans analyzed by RNA arbitrarily primed polymerase chain reaction.

Acidithiobacillus ferrooxidans is a chemoautotrophic bacterium that plays an important role in metal bioleaching processes. Despite the high level of tolerance to heavy metals shown by A. ferrooxidans, the genetic basis of copper resistance in this species remains unknown. We investigated the gene expression in response to copper in A. ferrooxidans LR using RNA arbitrarily primed polymerase chain reaction (RAP-PCR). One hundred and four differentially expressed genes were identified using eight arbitrary primers. Differential gene expression was confirmed by DNA slot blot hybridization, and approximately 70% of the RAP-PCR products were positive. The RAP-PCR products that presented the highest levels of induction or repression were cloned, sequenced and the sequences were compared with those in databases using the BLAST search algorithm. Seventeen sequences were obtained. The RAP-PCR product with the highest induction ratio showed similarity with the A. ferrooxidans cytochrome c. A high similarity with the thiamin biosynthesis gene thiC from Caulobacter crescentus was observed for another RAP-PCR product induced by copper. An RAP-PCR product repressed by copper showed significant similarity with the carboxysome operon that includes the ribulose-1,5-bisphosphate carboxylase/oxygenase complex from A. ferrooxidans and another copper-repressed product was significantly similar to the XyIN outer membrane protein from Pseudomonas putida. Finally, RAP-PCR products of unknown similarities were also present.

Base Sequence↗

PubMed2000

Differential gene expression in synovium of rheumatoid arthritis and osteoarthritis.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are the major types of arthritis. Although both diseases are characterized by joint destruction, their etiologies are different. To get insights into pathophysiological pathways, we used the suppression subtractive hybridization (SSH) method to identify differentially expressed genes in RA. DNA sequencing identified 12 gene products including cytoskeletal gamma-actin and extracellular matrix components such as fibronectin, collagen III alpha(1), and superficial zone protein. Interferon gamma-inducible genes such as a novel thiol reductase, two genes of unknown function (HSIFNIN4, RING3), and annexin II were also found. Two genes encoded proteins involved in proliferation such as elongation factor 1 alpha and the granulin precursor. Furthermore, the protease cathepsin B and synovial phospholipase A2 group IIA were detected by SSH. To confirm the differential expression of the genes, we performed RT-PCR analyses of RA and OA synovial tissues. Compared to OA patients, 9 of the 12 genes were overexpressed in RA, suggesting that SSH is a powerful tool for the detection of differential gene expression in synovial tissues. Further characterization of the gene products may help to identify pathophysiological mechanisms in arthritic diseases.

Adult↗

PubMed2006

Functional analysis of genes differentially expressed in the Drosophila wing disc: role of transcripts enriched in the wing region.

Differential gene expression is the major mechanism underlying the development of specific body regions. Here we assessed the role of genes differentially expressed in the Drosophila wing imaginal disc, which gives rise to two distinct adult structures: the body wall and the wing. Reverse genetics was used to test the function of uncharacterized genes first identified in a microarray screen as having high levels of expression in the presumptive wing. Such genes could participate in elaborating the specific morphological characteristics of the wing. The activity of the genes was modulated using misexpression and RNAi-mediated silencing. Misexpression of eight of nine genes tested caused phenotypes. Of 12 genes tested, 10 showed effective silencing with RNAi transgenes, but only 3 of these had resulting phenotypes. The wing phenotypes resulting from RNAi suggest that CG8780 is involved in patterning the veins in the proximal region of the wing blade and that CG17278 and CG30069 are required for adhesion of wing surfaces. Venation and apposition of the wing surfaces are processes specific to wing development providing a correlation between the expression and function of these genes. The results show that a combination of expression profiling and tissue-specific gene silencing has the potential to identify new genes involved in wing development and hence to contribute to our understanding of this process. However, there are both technical and biological limitations to this approach, including the efficacy of RNAi and the role that gene redundancy may play in masking phenotypes.

Amino Acid Sequence↗

PubMed2004

Screening for differential gene expression during the development of form-deprivation myopia in the chicken.

PURPOSE: To use the technique of differential gene display to analyze changes in gene expression that occur during the development of and recovery from form-deprivation myopia. METHODS: The differential display-reverse transcriptase-polymerase chain reaction technique was used to detect cDNAs that are differentially expressed after 24 h (including 12 h in the light) after fitting with a diffuser to induce form-deprivation myopia. Messenger RNA levels were determined by quantitative Northern blotting in retinas after 11 days of form deprivation or in retinas where the diffusers had been removed the previous day. RESULTS: Twenty-six differentially expressed genes were processed in our initial screen. Two of these, alphaB-crystallin and retinoic acid receptor-alpha, were studied further. Levels of alphaB-crystallin mRNA were increased on day 11 in retinas from form-deprived eyes relative to eyes of control chickens and were reduced to below those levels within 6 to 12 h after removal of the diffusers. Levels of retinoic acid receptor-alpha mRNA showed similar changes, except that after removal of the diffusers, the levels further increased. CONCLUSIONS: The technique of differential gene display can be used to detect changes in gene expression during the regulation of eye growth. The response of alphaB-crystallin is particularly interesting because expression increases when eye growth is high and decreases when eye growth slows.

Aging↗

PubMed2002

Screening for differentially expressed genes in the rat inner retina and optic nerve after optic nerve crush.

Limited optic nerve crush is a model of diffuse mechanical axon injury, the most prevalent cause of secondary neurodegeneration after closed head neurotrauma. In this report, a protocol is presented which allows for the rapid screening of differential gene expression in the inner retina, as well as the optic nerve, in response to partial nerve crush. To prove the reliability of the method, prototypically, the differential expression profiles of three candidate genes (kinesin light chain, ferritin, RYB-A) were verified. The method seems to be suitable to address the question of how differential gene expression contributes to degeneration, survival and axonal repair after partial nerve crush.

Animals↗

PubMed2006

Differential gene expression in response to ventricular unloading in rat and human myocardium.

UNLABELLED: Left ventricular unloading by mechanical assist devices induces myocardial atrophy. We aimed to systematically identify differentially expressed genes in a model of physiological atrophy (unloading of healthy rat myocardium) and compare these changes to those in a unloaded, failing human heart. METHODS: Atrophy in rat hearts was induced by heterotopic transplantation of a donor heart into the abdomen of an isogenic recipient. After one week, donor and recipient RNA was isolated. Differential gene expression was assessed by subtractive hybridization. Two screens with radioactive probes were performed to verify differentially expressed clones. Positive clones were sequenced and cDNA of genes of known homology were used as probes for hybridization with RNA from separate atrophied rat hearts and human tissue from a normal, failing or failing and unloaded left ventricle. RESULTS: We picked 1880 clones from the subtractive hybridization procedure (940/940: forward/reverse runs assessing up- or down-regulation, respectively). The first screen verified 465/140 and the second screen verified 67/30 clones. 24/23 clones were sequenced and 14/10 homologies to known genes were found. In the atrophied heart, respiratory chain and metabolic genes were down-regulated (NADH-DH, cytochrome c oxidase, acetyl-CoA synthetase, myoglobin) and cellular recognition and stress genes were up-regulated (MHC1 and 2, HSP70). In the human heart, cytochrome c oxidase, acetyl-CoA synthetase, and myoglobin expression was increased in the failing heart and returned to normal with unloading. Unloading also resulted in up-regulation of HSP70. CONCLUSIONS: The genetic responses of failing human and healthy rat myocardium to mechanical unloading show similarities that appear to be independent of species differences and/or underlying disease. Thus, heterotopic heart transplantation is a relevant model for investigating the mechanisms of mechanical unloading.

Animals↗

PubMed2002

Assessment of differential gene expression in human peripheral nerve injury.

BACKGROUND: Microarray technology is a powerful methodology for identifying differentially expressed genes. However, when thousands of genes in a microarray data set are evaluated simultaneously by fold changes and significance tests, the probability of detecting false positives rises sharply. In this first microarray study of brachial plexus injury, we applied and compared the performance of two recently proposed algorithms for tackling this multiple testing problem, Significance Analysis of Microarrays (SAM) and Westfall and Young step down adjusted p values, as well as t-statistics and Welch statistics, in specifying differential gene expression under different biological states. RESULTS: Using SAM based on t statistics, we identified 73 significant genes, which fall into different functional categories, such as cytokines / neurotrophin, myelin function and signal transduction. Interestingly, all but one gene were down-regulated in the patients. Using Welch statistics in conjunction with SAM, we identified an additional set of up-regulated genes, several of which are engaged in transcription and translation regulation. In contrast, the Westfall and Young algorithm identified only one gene using a conventional significance level of 0.05. CONCLUSION: In coping with multiple testing problems, Family-wise type I error rate (FWER) and false discovery rate (FDR) are different expressions of Type I error rates. The Westfall and Young algorithm controls FWER. In the context of this microarray study, it is, seemingly, too conservative. In contrast, SAM, by controlling FDR, provides a promising alternative. In this instance, genes selected by SAM were shown to be biologically meaningful.

Journal Article↗