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Results for “differential gene expression”

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PubMed2006

Subtractive differential display: a modified differential display technique for isolating differentially expressed genes.

Differential display (DD) is a novel PCR-based technique, very commonly used to study differentially expressed genes at the mRNA level. In this paper we report a modified version of this technique that we have used to study the differences between the mRNA population from brain tissue of adult and old rats. We have modified the technique to enhance reproducibility and reduce false positives and redundancy. It is fast and does not require any expensive or uncommon reagent. We choose to call it as subtractive differential display as it is a differential display performed over subtracted mRNA population. We have used this protocol successfully to clone a number of age-related differentially expressed sequences from rat brain that need to be sequenced to establish the gene identity.

Animals↗

PubMed2002

Identification of differentially expressed genes during cyclic adenosine monophosphate-induced neuroendocrine differentiation in the human prostatic adenocarcinoma cell line LNCaP.

The LNCaP cell line is a versatile and useful model suitable for the study of human prostate cancer in vitro. It has been determined that the elevation of LNCaP intracellular cyclic adenosine monophosphate (cAMP) levels through the addition of membrane-permeable cAMP analogs, phosphodiesterase inhibitors, adenylate cyclase activators, or components of the cAMP signal-transduction pathway can induce reversible neuroendocrine (NE) differentiation. Elucidation of those genes that are differentially expressed between undifferentiated prostate cancer cells and prostate cancer cells that have been induced to differentiate may present new insights into the molecular mechanisms governing NE differentiation, early detection of prostate cancer, and potential targets for gene therapy. In this study, differential display polymerase chain reaction (PCR) was used to identify 226 differentially expressed PCR products. Twelve of the differential display PCR products were confirmed by Northern blot analysis and were cloned. DNA sequencing and database comparisons were performed. To our knowledge, this is the first report of the use of differential gene expression techniques to analyze gene expression during cAMP-induced NE differentiation in LNCaP cells. Confirmation of NE differentiation reversibility also was accomplished.

Adenocarcinoma↗

PubMed1995

Analysis of differential gene expression in the kidney by differential cDNA screening, subtractive cloning, and mRNA differential display.

It is becoming increasingly evident that significant changes in gene expression occur during the course of disease progression in both genetic and nongenetic kidney diseases. Knowledge of the differentially expressed genes may yield information about the abnormal biochemical events that occur in the initiation and pathogenesis of these diseases. The purpose of this review is to provide an overview of some of the current approaches for identifying and analyzing differentially expressed genes. The power of these techniques lies in their their ability to detect differences in the levels of specific mRNAs in the diseased compared to the nondiseased kidney without prior knowledge of their identity. The three basic techniques considered are differential cDNA library screening, subtracted cDNA libraries, and PCR-based differential display. Emphasis is placed on cDNA library construction and differential screening. Also reviewed are the analysis of differentially expressed cDNAs by Southern and Northern blot hybridization, S1-protection, RT-PCR, DNA sequencing, and DNA sequence analysis.

Base Sequence↗

PubMed2000

Large-scale serial analysis of gene expression reveals genes differentially expressed in ovarian cancer.

Difficulties in the detection, diagnosis, and treatment of ovarian cancer result in an overall low survival rate of women with this disease. A better understanding of the pathways involved in ovarian tumorigenesis will likely provide new targets for early and effective intervention. Here, we have used serial analysis of gene expression (SAGE) to generate global gene expression profiles from various ovarian cell lines and tissues, including primary cancers, ovarian surface epithelia cells, and cystadenoma cells. The profiles were used to compare overall patterns of gene expression and to identify differentially expressed genes. We have sequenced a total of 385,000 tags, yielding >56,000 genes expressed in 10 different libraries derived from ovarian tissues. In general, ovarian cancer cell lines showed relatively high levels of similarity to libraries from other cancer cell lines, regardless of the tissue of origin (ovarian or colon), indicating that these lines had lost many of their tissue-specific expression patterns. In contrast, immortalized ovarian surface epithelia and ovarian cystadenoma cells showed much higher similarity to primary ovarian carcinomas than to primary colon carcinomas. Primary tissue specimens therefore appeared to be a better model for gene expression analyses. Using the expression profiles described above and stringent selection criteria, we have identified a number of genes highly differentially expressed between nontransformed ovarian epithelia and ovarian carcinomas. Some of the genes identified are already known to be overexpressed in ovarian cancer, but several represent novel candidates. Many of the genes up-regulated in ovarian cancer represent surface or secreted proteins such as claudin-3 and -4, HE4, mucin-1, epithelial cellular adhesion molecule, and mesothelin. Interestingly, both apolipoprotein E (ApoE) and ApoJ, two proteins involved in lipid homeostasis, are among the genes highly up-regulated in ovarian cancer. Selected serial analysis of gene expression results were further validated through immunohistochemical analysis of ApoJ, claudin-3, claudin-4, and epithelial cellular adhesion molecule in archival material. These experiments provided additional evidence of the relevance of our findings in vivo. The publicly available expression data reported here should stimulate and aid further research in the field of ovarian cancer.

Cell Transformation, Neoplastic↗

PubMed2001

[Differential gene expression in culturable and non-culturable Salmonella typhimurium].

Differential gene expression in culturable and non-culturable forms of Salmonella typhimurium was studied by the molecular display method. Six fragments of differentially expressed gene cDNA, depending on culturable or non-culturable state of the cultures, were isolated, cloned, and sequenced. Identification of corresponding S. typhimurium differentially expressed genes was carried out by comparing the sequences of cDNA fragments with the bacterial genome data base.

DNA Primers↗

PubMed2002

Integration of amplified differential gene expression (ADGE) and DNA microarray.

Amplified Differential Gene Expression (ADGE) provides a new concept that the ratios of differentially expressed genes are magnified before detection in order to improve both sensitivity and accuracy. This technology is now implemented with integration of DNA reassociation and PCR. The ADGE technique can be used either as a stand-alone method or in series with DNA microarray. ADGE is used in sample preprocessing and DNA microarray is used as a displaying system in the series combination. These two techniques are mutually synergistic: the quadratic magnification of ratios of differential gene expression achieved by ADGE improves the detection sensitivity and accuracy; the PCR amplification of templates enhances the signal intensity and reduces the requirement for large amounts of starting material; the high throughput for DNA microarray is maintained.

Algorithms↗

PubMed2006

Detection of differential gene expression in brown adipose tissue of hibernating arctic ground squirrels with mouse microarrays.

Hibernation is an energy-saving strategy adopted by a wide range of mammals to survive highly seasonal or unpredictable environments. Arctic ground squirrels living in Alaska provide an extreme example, with 6- to 9-mo-long hibernation seasons when body temperature alternates between levels near 0 degrees C during torpor and 37 degrees C during arousal episodes. Heat production during hibernation is provided, in part, by nonshivering thermogenesis that occurs in large deposits of brown adipose tissue (BAT). BAT is active at tissue temperatures from 0 to 37 degrees C during rewarming and continuously at near 0 degrees C during torpor in subfreezing conditions. Despite its crucial role in hibernation, the global gene expression patterns in BAT during hibernation compared with the nonhibernation season remain largely unknown. We report a large-scale study of differential gene expression in BAT between winter hibernating and summer active arctic ground squirrels using mouse microarrays. Selected differentially expressed genes identified on the arrays were validated by quantitative real-time PCR using ground squirrel specific primers. Our results show that the mRNA levels of the genes involved in nearly every step of the biochemical pathway leading to nonshivering thermogenesis are significantly increased in BAT during hibernation, whereas those of genes involved in protein biosynthesis are significantly decreased compared with summer active animals in August. Surprisingly, the differentially expressed genes also include adipocyte differentiation-related protein or adipophilin (Adfp), gap junction protein 1 (Gja1), and secreted protein acidic and cysteine-rich (Sparc), which may play a role in enhancing thermogenesis at low tissue temperatures in BAT.

Adipose Tissue, Brown↗

PubMed2004

Identifying differentially expressed genes from microarray experiments via statistic synthesis.

MOTIVATION: A common objective of microarray experiments is the detection of differential gene expression between samples obtained under different conditions. The task of identifying differentially expressed genes consists of two aspects: ranking and selection. Numerous statistics have been proposed to rank genes in order of evidence for differential expression. However, no one statistic is universally optimal and there is seldom any basis or guidance that can direct toward a particular statistic of choice. RESULTS: Our new approach, which addresses both ranking and selection of differentially expressed genes, integrates differing statistics via a distance synthesis scheme. Using a set of (Affymetrix) spike-in datasets, in which differentially expressed genes are known, we demonstrate that our method compares favorably with the best individual statistics, while achieving robustness properties lacked by the individual statistics. We further evaluate performance on one other microarray study.

Algorithms↗

PubMed1988

Differentiation-induced gene expression in 3T3-L1 preadipocytes. Characterization of a differentially expressed gene encoding stearoyl-CoA desaturase.

Previous studies have shown that differentiation of 3T3-L1 preadipocytes leads to the activation of transcription of an unidentified gene which encodes a 4.9-kilobase (kb) mRNA. Several cDNAs that include the complete sequence of this mRNA were obtained and used to isolate and characterize the gene. Analysis of the nucleotide and amino acid sequences of both cDNA and genomic clones revealed that the gene encodes the mouse stearoyl-CoA desaturase (SCD), an enzyme known to be expressed upon differentiation of 3T3-L1 preadipocytes. The predicted amino acid sequence (355 residues) of the mouse 3T3-L1 adipocyte SCD exhibits 92% identity to that of the rat liver SCD. There is also a high degree of nucleotide sequence identity between the mouse and rat mRNAs in their unusually long approximately 3.5-kb 3'-untranslated regions. Mice fed a diet containing unsaturated triacylglycerides express SCD mRNA only in adipose tissue, whereas mice starved and refed a fat-free diet, express SCD mRNA in both liver and adipose tissue. The mouse gene for the desaturase spans approximately 15 kb and contains 6 exons and 5 introns with all intron-exon junctions conforming to the GT/AG splicing rule. As determined by S1 nuclease mapping and primer extension analysis, the transcriptional initiation site maps 152 nucleotides upstream from the initiation methionine codon. A canonical promoter "TATA" box is located 30 base pairs upstream of the Cap site. A typical "CCAAT" box sequence is not present in the adjacent 5'-flanking region; however, there is a GC-rich sequence (at nucleotide -215) similar to the binding site for the nuclear transcription factor Sp1. Upstream from the transcriptional initiation site are elements with homology (approximately 75%) to the putative fat-specific transcriptional element FSE2 and core consensus sequences for cAMP and glucocorticoid regulatory elements. A chimeric construct, containing 363 base pairs of 5'-flanking sequence and 30 nucleotides of 5'-untranslated sequence of the mouse SCD gene ligated to the bacterial chloramphenicol acetyltransferase gene, was transfected into 3T3-L1 cells. When cells were induced to differentiate into adipocytes, expression of the SCD chloramphenicol acetyltransferase gene increased approximately 63-fold, suggesting that the SCD promoter region contains elements that mediate the response to adipogenic agents which induce differentiation.

Adipose Tissue↗

PubMed1999

Oxidative stress induces differential gene expression in a human lens epithelial cell line.

PURPOSE: To identify differentially expressed genes in a human lens epithelial cell line exposed to oxidative stress. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) differential display was used to evaluate differential gene expression in a human lens epithelial cell line (SRA 01-04) when cells were exposed for 3 hours to a single bolus of 200 microM hydrogen peroxide. Differentially expressed genes were identified through DNA sequencing and a nucleotide database search. Differential expression was confirmed by northern blot and RT-PCR analyses. RESULTS: Using 18 primer sets, 28 RT-PCR products were differentially expressed between control and hydrogen peroxide-treated cells. In stressed cells, mitochondrial transcripts nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4 and cytochrome b were downregulated 4-fold. Of the cytoplasmic mRNAs, glutamine cyclotransferase decreased 10-fold, whereas cytokine-inducible nuclear protein, alternative splicing factor 2, and beta-hydroxyisobutyryl-coenzyme A hydrolase increased 2-, 4-, and 10-fold, respectively. Analysis of mitochondrial transcripts in a 24-hour time course showed that NADH dehydrogenase subunit 4 mRNA decreased by 2-fold as early as 1 hour after oxidative stress, whereas the rate of decrease was slower for cytochrome b, cytochrome oxidase III, and 16S rRNA. CONCLUSIONS: Oxidative stress induced specific expressed gene changes in hydrogen peroxide-treated lens cells, including genes involved in cellular respiration and mRNA and peptide processing. These early changes may reflect pathways involved in the defense, pathology, or both of the lens epithelium, which is exposed to oxidative stress throughout life.

Blotting, Northern↗

PubMed2004

Differential gene expression during interactions between Heterobasidion annosum and Physisporinus sanguinolentus.

Using mRNA differential display we have identified differentially expressed genes in non-self-interacting vs. single mycelia of the conifer pathogen Heterobasidion annosum and the wood decomposing basidiomycete Physisporinus sanguinolentus. Altogether 39 differentially displayed bands were cloned and sequenced, corresponding to 21 unique genes, which were confirmed by semi-quantitative RT-PCR to be differentially expressed. Further confirmation of differential gene expression was made by real time RT-PCR. All 10 genes identified from P. sanguinolentus had lower expression, while in H. annosum three genes had higher and eight lower expression in non-self-interacting mycelia vs. single mycelia. One of the induced genes showed high similarity to the Coprinus cinereus recA/RAD51 homolog (rah1) which is essential for homologous recombination, DNA repair and stress responses.

Basidiomycota↗

PubMed2003

Novel differential gene expression in human cirrhosis detected by suppression subtractive hybridization.

Pathogenic molecular pathways in cirrhotic liver diseases such as hepatitis C virus (HCV), autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) are poorly characterized. Differentially expressed genes are often important in disease pathogenesis. Suppression subtractive hybridization (SSH) is a genome-wide approach that enriches for differentially expressed mRNA transcripts. We aimed to make novel observations of differential gene expression in cirrhosis using SSH combined with quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Liver transcriptomes in HCV cirrhosis, AIH cirrhosis, PBC, and nondiseased liver tissue were examined by SSH. Resulting complementary DNA (cDNA) clones were rescreened for differential expression by dot-blot hybridization and then sequenced. Selected gene expression was quantified by real-time RT-PCR. Following SSH, 694 clones were rescreened for differential gene expression, of which 145 were sequenced and found to derive from 89 different genes. Seven clones were homologous only with expressed sequence tag (EST) sequences encoding genes having no known function. Up-regulated expression of four genes was confirmed by real-time RT-PCR: transmembrane 4 superfamily member 3 (tetraspanin CO-029) in all forms of cirrhosis, hedgehog interacting protein (HIP) in AIH cirrhosis and chitinase 3-like-1 (HC gp-39 or ykl-40) and arginine-glutamic acid repeat (RERE) in HCV cirrhosis. RERE gene polymorphisms and splice variants were observed in all tissues examined. Tetraspanin CO-029 up-regulation was primarily localized to bile ductular cells. In conclusion, novel observations of differential gene expression in human cirrhosis were made using SSH as the primary discovery tool. In particular, further studies of the RERE gene and its products in HCV associated liver disease are warranted.

Adipokines↗

PubMed1995

Differential display of messenger ribonucleic acid: a useful technique for analyzing differential gene expression in human brain tumors.

In this study, a differential display method for messenger ribonucleic acid was successfully used to identify genes differentially expressed between normal human brain and malignant glioma tissues. A total of 60 differentially expressed sequences were initially identified, of which 21 were cloned and sequenced. Twenty of the cloned sequences represented novel genes, and one sequence represented a kinesin heavy chain (KHC) gene isoform. The KHC isoform was selected for further characterization. Northern blots of total ribonucleic acid isolated from normal brain and a glioblastoma were probed with our KHC probe and confirmed the differential expression of this gene. Expression analysis of a variety of normal human tissues demonstrated that this KHC isoform is expressed only in brain tissues, with no detectable expression in placenta, spleen, kidney, lung, liver, or skeletal muscle. Our results confirm the rapid and sensitive nature of the differential display technique in identifying differential gene expression. This method offers a means to identify new genes of biological interest in human brain tumors such as oncogenes, tumor suppressor genes, and tumor-specific markers.

Adult↗

PubMed2003

Expression analysis of subtractively enriched libraries (EASEL): a widely applicable approach to the identification of differentially expressed genes.

A variety of methods for high throughput analysis of differential gene expression has been developed over the past years. We have implemented the EASEL technique that adds flexibility, efficiency and wide-applicability to these methods. The EASEL procedure is unique as it integrates several well established techniques and thereby offers a combination of subtractive hybridization of 3' cDNA ends with macroarrays analysis and Serial Analysis of Gene Expression (SAGE). In addition, once a set of interesting, differentially expressed genes is identified, the material required for follow up studies to test the hypothesis that the gene is truly involved in the process of interest is readily available. In this report, we first present a step-by-step validation of the procedure, since several of the incorporated steps had to be tailored to meet specific requirements and implied drastic modifications of the original methods. Secondly, we applied EASEL to the identification of up-regulated gene products in the outflow tract region of the embryonic rat heart. Here we provide evidence that at least two among the differentially expressed genes detected, follistatin-like protein gene and membrane type 1-metallo proteinase gene, are selectively up-regulated in the outflow tract, suggesting their involvement in the development of this region during embryogenesis.

Animals↗

PubMed1999

A novel strategy for identifying differential gene expression: An improved method of differential display analysis.

We propose a novel alternative approach, an advanced method for recently developed strategies, for identifying differentially expressed genes. Firstly, double-stranded cDNAs were digested using Sau3AI and the 3'-end restriction fragments of the cDNA were ligated to a double-stranded adapter. Next, the restriction fragments were directly amplified using several combinations of adapter-specific primers and FITC-labeled oligo dT primers. The selected cDNA fragments were displayed on a polyacrylamide gel. Neither nested PCR nor purification of 3'-end fragments are necessary. We examined the validity of this approach by evaluating gene expression changes during granulocytic differentiation of HL-60 cells. This method can theoretically detect almost all gene expression changes more rapidly and through simpler manipulations than by any other approach.

Cell Differentiation↗

PubMed1999

Differential gene expression in retina of myopic chicken eyes using mRNA differential display.

PURPOSE: To study differentially expressed genes in retina of experimental myopic chicken. METHODS: Experimental myopia in chicken was induced by form-deprivation. The mRNA in chicken retina was analyzed by using differential display. RESULTS: Experimental myopia was successfully induced in chicken through form-deprivation. Differentially expressed gene fragments were detected in retina of chicken with myopic development and recovery as compared with normal controls. CONCLUSION: The differential display of mRNA may be a useful way in cloning myopic-related genes.

Animals↗

PubMed2001

Differential gene expression during capillary morphogenesis in 3D collagen matrices: regulated expression of genes involved in basement membrane matrix assembly, cell cycle progression, cellular differentiation and G-protein signaling.

We have performed a screening analysis of differential gene expression using a defined in vitro model of human capillary tube formation. Gene array, differential display and cDNA library screening were used to identify both known and novel differentially expressed genes. Major findings include: the upregulation and functional importance of genes associated with basement membrane matrix assembly; the upregulation of growth factors, transcription factors, anti-apoptotic factors, markers of endothelial cell differentiation, JAK-STAT signalling molecules, adhesion receptors, proteinase inhibitors and actin regulatory proteins; and expression changes consistent with inhibition of cell cycle progression, increased cholesterol biosynthesis, decreased ubiquitin-proteasome mediated degradation, and activation of G-protein signaling pathways. Using DNA microarray analysis, the most induced genes at 8, 24 and 48 hours compared with those at 0 hours were jagged-1, stanniocalcin and angiopoietin-2, whereas the most repressed genes were connective tissue growth factor, fibulin-3 and RGS-5. In addition, the full length coding sequence of two novel regulated capillary morphogenesis genes (CMGs) are presented. CMG-1 encodes a predicted intracellular 65 kDa protein with coiled-coil domains. A CMG-1-green fluorescent protein (GFP) chimera was observed to target to an intracellular vesicular compartment. A second novel gene, CMG-2, was found to encode a predicted intracellular protein of 45 kDa containing a transmembrane segment and a CMG-2-GFP chimera was observed to target to the endoplasmic reticulum. A recombinant portion of CMG-2 was found to bind collagen type IV and laminin, suggesting a potential role in basement membrane matrix synthesis and assembly. These data further elucidate the genetic events regulating capillary tube formation in a 3D matrix environment.

Amino Acid Sequence↗

PubMed2006

Probe-level linear model fitting and mixture modeling results in high accuracy detection of differential gene expression.

BACKGROUND: The identification of differentially expressed genes (DEGs) from Affymetrix GeneChips arrays is currently done by first computing expression levels from the low-level probe intensities, then deriving significance by comparing these expression levels between conditions. The proposed PL-LM (Probe-Level Linear Model) method implements a linear model applied on the probe-level data to directly estimate the treatment effect. A finite mixture of Gaussian components is then used to identify DEGs using the coefficients estimated by the linear model. This approach can readily be applied to experimental design with or without replication. RESULTS: On a wholly defined dataset, the PL-LM method was able to identify 75% of the differentially expressed genes within 10% of false positives. This accuracy was achieved both using the three replicates per conditions available in the dataset and using only one replicate per condition. CONCLUSION: The method achieves, on this dataset, a higher accuracy than the best set of tools identified by the authors of the dataset, and does so using only one replicate per condition.

Algorithms↗