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PubMed1998

A simple and efficient method for the isolation of differentially expressed genes.

A simple and reproducible general approach for the isolation of differentially expressed genes is described. Digestion of cDNAs with a class IIs restriction endonuclease produces fragments with every combination of possible bases in the cohesive ends. Under stringent conditions, the specific ligation of adaptors with perfectly complementary overhangs partitions the cDNA fragments into non-overlapping subpopulations. Internal cDNA restriction fragments are exponentially amplified by adaptor primer PCR and visualised by non-denaturing polyacrylamide gel electrophoresis. The power of the technology was demonstrated using a rat model of pressure-induced left-ventricular hypertrophy (LVH). A set of 29 fragments, derived from a sample (6 %) of the possible adaptor pool combinations, displayed apparent differential expression. The differential expression of 19 (66 %) were confirmed by Northern blot analysis. Sequence analysis identified both genes known to be upregulated in LVH, and novel genes. The fidelity of adaptor ligation was demonstrated by the isolation of known gene fragments by appropriate adaptor combinations. The spiking of mRNA populations with known amounts of a synthetic mRNA demonstrated a current sensitivity equivalent to the detection of transcripts expressed at the level of as little as 1 in 10,000 molecules.

Animals↗
PubMed2006

[cDNA microarray-based screening of differentially expressed genes in macrophages in the spleen of patients with portal hypertension and hypersplenism].

OBJECTIVE: To identify the differentially expressed genes associated with hypersplenism in patients with portal hypertension. METHODS: The total RNA were extracted from the macrophages isolated from normal spleen and the spleen of patients with portal hypertension and reversely transcribed to cDNA with the incorporation of fluorescent (cy3 and cy5)-labeled dCTP to prepare the hybridization probes. After hybridization of Biostar-H140s chip containing 14,112 spots of cDNAs with the prepared probes, the gene chip was scanned for fluorescence intensity to screen the differently expressed genes. Three gene chips were used for hybridization and only the genes with differential expression in all the three chips were considered to associate with hypersplenism in patients with portal hypertension. RESULTS: Totaling 896, 1330 and 898 genes were identified to be differentially expressed by the three chips, respectively, and 121 genes (0.86%) showed differential expression in all the three chips, including 21 up-regulated known genes and 73 down-regulated known genes. The differently expressed genes were functionally related with ion channels and transport proteins, cyclins, cytoskeleton, cell receptors, cell signal transduction, metabolism, immunity, and so forth. These genes might be involved in hypersplenism in the condition of portal hypertension. CONCLUSION: cDNA microarray-based screening of differentially expressed genes in the macrophages in the spleen may provide new insights into the pathogenesis of hypersplenism in patients with portal hypertension.

Female↗
PubMed2003

Isolation and characterization of a cDNA from Quercus robur differentially expressed in juvenile-like and mature shoots.

A full-length cDNA clone named QRCPE (Quercus robur crown preferentially expressed) that is differentially expressed during in vitro culture of mature and juvenile-like shoots of Quercus robur L. was identified by differential display. The deduced amino acid sequence showed that the encoded protein is small, contains a predicted N-terminal hydrophobic signal peptide that targets the protein to the cell wall, and is rich in glycine and histidine residues. Accumulation of QRCPE mRNA was higher in oak microshoots derived from crown branch shoot cultures than in oak microshoots derived from basal shoot cultures at the end of the multiplication and rooting period. Among organs, the highest accumulation of QRCPE transcripts was detected in roots, followed by stems and leaves, with preferential accumulation in specific organs of ontogenetically older shoots. Although QRCPE mRNA was abundant in oak zygotic and somatic embryos, almost no QRCPE mRNA accumulation was detected in nodular callus cells, suggesting a possible role of this gene in embryonic development. In proliferating shoot cultures of two chestnut (Castanea sativa Mill.) clones, the QRCPE homolog was preferentially expressed in crown-derived shoots. On the other hand, expression analysis of QRCPE in juvenile and mature material from soil-grown oak plants indicated that this gene is expressed from the embryonic to mature phases, but is progressively down-regulated during plant maturation. In vitro culture conditions induced changes in QRCPE transcript abundance in both basal and crown shoots in a phase-dependent manner. We conclude that QRCPE expression in oak is correlated with the ontogenetic stage of shoots, and thus this gene may be useful as a potential molecular marker for maturation-related characteristics.

Aesculus↗
PubMed2005

[Detection of apoptosis genes differentially expressed in patients with different degrees of benzene poisoning by cDNA microarray].

OBJECTIVE: To investigate the differential expression of apoptosis genes in patients with different degrees of benzene poisoning by cDNA microarray. METHODS: Peripheral mononuclear cells were isolated from seven patients with benzene poisoning of different degrees (suspected 1 case, moderate 2, severe 2), and seven age and sex matched normal control subjects. Total RNA was extracted and purified, followed by reverse transcription to cDNAs with concomitant incorporation of fluorescent dCTP (Cy3 or Cy5). Then 177 genes associated with cell apoptosis were hybridized against the cDNAs probes in microarray. Fluorescent signals were scanned to detect apoptosis genes differentially expressed in patients and normal subjects. RESULTS: Forty one genes were found to be differentially expressed between benzene-poisoned patients and normal controls; among the 41 genes, three were up-regulated among patients with mild to moderate degrees of benzene poisoning and one up-regulated among all patients. The total amount of differentially expressed genes of apoptosis decreased with the aggravation of benzene poisoning. CONCLUSIONS: Differential expression of apoptosis genes was found in patients with benzene poisoning, suggesting a role of altered apoptosis in benzene-induced hematotoxicity.

Apoptosis↗
PubMed1999

Identification of differentially expressed mRNAs in human fetal liver across gestation.

Differential gene expression, with its precise start and stop times, is believed to be critical for the programmed development of new cells and tissues. Within the developing fetus, one tissue of particular interest is fetal liver. This organ undergoes rapid changes in the pathway toward liver development in utero since it is also the major site of hematopoiesis, until bone marrow hematopoiesis predominates. Believing that patterns would emerge from the bi-weekly large-scale inspection of expressed genes in the fetal liver, we employed differential display reverse transcription-polymerase chain reaction (DDRT-PCR) as ourprimary inspection tool. Using DDRT-PCR, we isolated cDNAs differentially expressed throughout fetal liver development and in adult liver. We displayed approximately 25 000 cDNAs from 10 and 24 week fetal liver and adult liver. From this initial screen, we determined that approximately 0.1-1% of the mRNA population undergoes expression changes. We extracted, purified and sequenced 25 differentially displayed cDNA bands. Fourteen cDNAs had similarities to known genes, while 11 cDNAs were not similar to any characterized gene. The differentially expressed cDNAs from known genes present in fetal liver include alpha-fetoprotein, stem cell factor, erythroid alpha-spectrin, 2,3-bisphosphoglycerate mutase, insulin-like growth factor-2, porphobilinogen deaminase and Mac30. The differentially expressed cDNAs present in adult liver but not in 10 week fetal liver were nicotinamide deaminase, human fibrinogen-related protein and alpha-acid glycoprotein. The majority of differentially expressed genes found during this effort appear to be turned on during organogenesis, however, some genes were found that are apparently turned off completely.

Adult↗
PubMed2003

Expression profiling and characterization of 4200 genes cloned from primary neuroblastomas: identification of 305 genes differentially expressed between favorable and unfavorable subsets.

Neuroblastoma (NBL), one of the most common childhood solid tumors, has a distinct nature in different prognostic subgroups: NBL in patients under 1 year of age usually regresses spontaneously, whereas that in patients over 1 year of age often grows aggressively and eventually kills the patient. To understand the molecular mechanism of biology and tumorigenesis of NBL, we decided to perform a comprehensive approach to unveil the gene expression profiles among the NBL subsets. We constructed the subset-specific oligo-capping cDNA libraries from the primary NBL tissues with favorable (F: stage 1, high expression of TrkA and a single copy of MYCN) and unfavorable (UF: stage 3 or 4, decreased expression of TrkA and MYCN amplification) characteristics and randomly cloned 4654 cDNAs. Among 4243 cDNAs sequenced successfully, 1799 (42.4%) were the genes with unknown function. Excluding the housekeeping genes, an expression profile of each subset was extremely different. To determine the genes expressed differentially between F and UF subsets, we performed semiquantitative reverse transcriptase (RT)-PCR for each of the 1842 independent genes using RNA obtained from 16 F and 16 UF NBLs as template. This revealed that 278 genes were highly expressed in the F subset as compared to the UF one, while, surprisingly, only 27 genes were expressed at higher levels in the UF rather than the F subset. These differentially expressed genes included 194 genes with unknown function. Many of the genes expressed at high levels in the F subset were related to catecholamine biosynthesis, small GTPases, synapse formation, synaptic vesicle transport, and transcription factors regulating differentiation of the neural crest-derived cells. On the other hand, the genes expressed at high levels in the UF subset included transcription factors and/or receptors that might regulate neuronal growth and differentiation. The chromosomal mapping of those genes showed some clusters. Thus, our mass-identification and characterization of the differentially expressed genes between the subsets may become a powerful tool for finding the important genes of NBL as well as developing new diagnostic and therapeutic strategies against aggressive NBL.

Chromosome Mapping↗
PubMed1992

Differential expression of guanine nucleotide-binding proteins enhances cAMP synthesis in regenerating rat liver.

Events leading to cAMP accumulation after partial hepatectomy (PH) and effects of cAMP on hormonal induction of DNA synthesis in hepatocytes were characterized. Hepatic cAMP peaked biphasically post-PH and paralleled changes in adenylyl cyclase activity. Fluctuations in cyclase activity were not explained by variations in glucagon receptor kinetics, but reflected altered G-protein expression. Membrane levels of the stimulatory G-protein, Gs alpha, increased early after PH and were sustained. Levels of the inhibitory G-protein, Gi2 alpha, increased more slowly, peaked later, and quickly fell. Levels of both G-proteins correlated poorly with levels of their mRNAs, suggesting posttranscriptional factors modify their membrane concentrations. When growth factor-induced DNA synthesis was compared in hepatocyte cultures grown with or without agents that increase intracellular cAMP, DNA synthesis was inhibited by sustained high levels of cAMP but was enhanced when high cAMP levels fell. In both regenerating liver and hepatocyte cultures, the expression of a "differentiated" hepatocyte gene, phosphoenolpyruvate carboxykinase, correlated with elevated cAMP levels. These data suggest that the differential expression of G-proteins integrates signals initiated by several growth factors so that the accumulation of cAMP is tightly regulated post-PH. The ensuing variations in cAMP levels modulate both growth and differentiated functions during liver regeneration.

Animals↗
PubMed2006

Comparative proteomic analysis reveals differential expression of Hsp25 following the directed differentiation of mouse embryonic stem cells.

Murine embryonic stem (ES) cells can be committed to neural differentiation with high efficiency in culture through the use of feeder- and serum-free media. This system is proving to be an excellent model to study processes involved in ES cell commitment to neural cell fate. We used this approach to generate neurogenic embryoid bodies (NEBs) in a serum-free culture system to perform proteomic analysis of soluble fractions and identify early changes in protein expression as ES cells differentiate. Ten candidate proteins were altered significantly in expression levels. One of the most significant alterations was for the small heat shock protein Hsp25. Three species of Hsp25 are detected in ES cells, and this expression pattern changes during the first 24 h of differentiation until expression is decreased to levels that are barely detectable at 4 days following differentiation. We used immunofluorescence studies to confirm that following ES cell differentiation, expression of Hsp25 becomes excluded from neural precursors as well as other differentiating cells, making it a potentially useful marker of early ES cell differentiation.

Animals↗
PubMed2002

Analysis of differentially expressed genes in hepatocellular carcinoma using cDNA arrays.

Hepatocellular carcinoma (HCC) is characterized by multiple somatic mutations, including DNA rearrangements, that affect many cell-growth regulatory pathways. Many genes differentially expressed in HCC have been reported previously, but the patterns of expression varied significantly between patients who bore different risk factors for HCC. To identify genes whose differential expression could serve as a "signature" for diagnosis and prognosis of HCC, we performed analyses of differentially expressed genes in three cases of HCC with different risk factors using the Atlas Human Cancer cDNA Expression Arrays. Among all 597 genes present on the array, only three were found to be coordinately differentially expressed in all three HCC cases, in agreement with published data. These three genes, Cu/Zn superoxide dismutase, osteonectin/secreted protein acidic and rich in cysteine, and matrix metalloproteinase 14, could serve as candidates for the HCC "signature." Ten genes were found to be coordinately differentially expressed in only two of three tested HCC cases. On the other hand, many genes that had been reported previously as differentially expressed in HCC failed to show the described pattern of expression in this group. The results of this study confirm the great variability in gene-expression patterns in HCC and establish the utility of the array technology for identifying both the HCC signature genes and individual gene-expression patterns for purposes of patient-oriented therapy.

Aged↗
PubMed2002

Isolation of novel differentially expressed genes related to human glioma using cDNA microarray and characterizations of two novel full-length genes.

Identification of the genes that are differentially expressed between brain tumor and normal brain tissues is important for understanding the molecular basis of these nerve system tumors and for defining possible targets for therapeutic intervention. This investigation is intended to obtain differentially expressed genes related to human glioma using cDNA microarray. Total RNA was extracted from human glioma specimens and normal brain tissues, and mRNA was obtained by oligotex chromatography. The cDNA microarray contains 4366 novel cDNA clones. The results of hybridization were scanned using computer system. Two genes selected from the results of cDNA microarray hybridization were subsequently analyzed by bio-informatic approach, Northern blot, in situ hybridization and radiation hybridization. We demonstrated that at a differentially expressed ration of two to three times, 15 cDNA clones were considered differentially expressed. Two novel full-length genes were selected for further investigation, one named human PKIbeta gene (clone 436F11, GenBank with accession number: AF225513) was over-expressed in normal brain tissues and the other named human ribosomal protein L14.22 gene (clone 507E08, GenBank with accession number: AF329277) was over-expressed in gliomas. Furthermore, the 436F11 gene was located on 6q21-q23 between the D6S304 and D6S2156 markers, while the 507E08 gene was located between the D14S1066 and D14S265 markers. We realized that cDNA microarray technology can be successfully applied to identify differentially expressed genes in human glioma. This approach is superior to routine representational difference analysis, suppression subtractive hybridization and Northern blot for detection and isolation of differentially expressed genes in different tissues. At present, we have discovered two novel full-length genes related to human glioma and their characterizations have been partially clarified.

Amino Acid Sequence↗
PubMed2003

[Isolation of differentially expressed genes in human osteoblast-like osteosarcoma cell line induced with 17beta-estradiol].

OBJECTIVE: To obtain a serial differentially expressed cDNA fragments from human osteoblast-like osteosarcoma MG-63 cells induced with 17beta-estradiol and to find some estrogen-responsive genes. METHODS: Optimized cDNA representational difference analysis (RDA) was performed to isolate up-regulated expressed sequences between cDNA from MG-63 cell line treated with and without 17beta-estradiol. The sources of up-regulated expressed cDNA fragments were proved by Southern blot. The fragments were cloned into the pGEM-T easy vector and a cDNA library was prepared in E. coli JM109 cells. The cDNA library was plated on LB/Amp(+)/X-gal/IPTG plates and white colonies were picked up and individually grown in LB/Amp(+) medium in 96-well plates. After PCR, colonies were individually blotted onto a Hybond N membrane. Membranes were hybridized with alpha-(32)P-labeled subtracted or unsubtracted tester cDNA. Clones showing a strong hybridization signal with the forward-subtracted probes compared with the reverse-subtracted ones were selected for DNA sequencing, BLAST and Northern blot analysis after release of the pGEM-T easy insert with EcoR I. RESULTS: Five up-regulated expressed fragments were isolated in the fourth subtraction hybridization using cDNA from MG-63 cells induced with 17beta-estradiol as tester amplicon and cDNA from untreated MG-63 cells as driver amplicon by cDNA RDA. These fragments were proved to be really coming from tester amplicon and down-regulated expressed in the untreated cell by Southern blotting. We obtained more than 600 cDNA clones with positive insert from MG-63 cells line induced with 17beta-estradiol and about 120 differentially expressed clones through dot blotting. Twenty clones were sequenced and we got 15 gene sequences. One of them was proved to be differentially expressed through Northern blotting. CONCLUSIONS: cDNA RDA is one of the most effective methods which can isolate differentially expressed genes and we can screen differentially expressed genes rapidly through cDNA RDA combined with cDNA arrays. Some genes of human osteoblast-like osteosarcoma MG-63 cell line showed up-regulated expression after induction by 17beta-estradiol.

Blotting, Northern↗
PubMed1995

Differential expression of the full-length and secreted truncated forms of EGF receptor during formation of dental tissues.

The developmental regulation of various receptor forms may be a key-element in the local fine tuning of growth factor effects. The present study focuses on the tissue- and stage-specificity of the alternative splicing of EGF receptor transcripts in the rat incisor. In situ hybridization, as well as light- and electron-microscopic immunolocalization were performed with a set of tools which enable us to discriminate the full-length and secreted truncated forms of EGF receptor. Our data show that, apart from a transient expression in differentiating odontoblasts, EGF receptor expression was predominantly observed in the dental epithelium. In the crown, the expression of the full-length EGF receptor was maximal during preameloblast proliferation and differentiation, decreased in differentiated ameloblasts, and remained low throughout enamel secretion. On the other hand, maturation stage ameloblasts, which regulate the final mineralization of enamel, express high levels of the full-length EGF receptor. In contrast with ameloblasts, epithelial supra-ameloblastic cells, which are not directly involved in the deposition of enamel matrix, showed an alternating predominance of the secreted truncated form during the secretion stage, and the full-length form during the maturation stage. The presence of the secreted truncated EGF receptor form was supported by the electron microscopic detection of extracellular aggregates of immunoreactive EGF receptor. Finally, Northern-blotting of enamel organ samples confirmed the presence of transcripts corresponding to mRNAs of both EGF receptor forms. During root formation, a decreasing gradient of full-length EGF receptor form expression was observed from the apical loop to the disrupting zone in root epithelium. The secreted truncated EGF receptor form was essentially detected in epithelial cells of the disrupting zone of root epithelium. During crown formation, the secreted truncated EGF receptor form, which appears to be synthesized by epithelial supra-ameloblastic cells and secreted toward ameloblasts, may competitively bind EGF receptor ligands and modify activation of the full-length EGF receptor.

Aging↗
PubMed2004

[Differentially expressed genes in prostate cancer and benign prostatic hyperplasia].

OBJECTIVE: To detect and analyze the differentially expressed genes in prostate cancer (PCa) and benign prostatic hyperplasia (BPH). METHODS: Oligonucleotide microarray containing 465 genes was used to investigate the differentially expressed genes in PCa and BPH. RESULTS: There were 35 differentially expressed genes between PCa and BPH, of which 17 were up-regulated and 18 down-regulated in PCa. CONCLUSION: The study of the differentially expressed genes in PCa and BPH should help to understand the molecular mechanism of PCa and identify the markers for diagnostic and therapeutic use.

Down-Regulation↗
PubMed2005

Rapid identification of differentially expressed genes by in situ screening of bacteria.

The identification of differentially expressed genes is a key step in the understanding of specific molecular mechanisms. Various methods have been developed to search for differences in expression but most of them are time or money consuming. We present here an alternative technique that connects standard suppression subtractive hybridization with in situ screening of bacteria to isolate and identify differentially expressed transcripts. The in situ differential screening is based on the transfer of bacteria directly from cultures onto nylon membranes with no need of phenol/chloroform extraction, colony lifting, or polymerase chain reaction amplification. This improved method was successfully applied and must be seen as a simple, low-cost, time-saving, and reproducible approach to identify differentially expressed genes.

Bacteria↗
PubMed2002

Nonparametric methods for identifying differentially expressed genes in microarray data.

MOTIVATION: Gene expression experiments provide a fast and systematic way to identify disease markers relevant to clinical care. In this study, we address the problem of robust identification of differentially expressed genes from microarray data. Differentially expressed genes, or discriminator genes, are genes with significantly different expression in two user-defined groups of microarray experiments. We compare three model-free approaches: (1). nonparametric t-test, (2). Wilcoxon (or Mann-Whitney) rank sum test, and (3). a heuristic method based on high Pearson correlation to a perfectly differentiating gene ('ideal discriminator method'). We systematically assess the performance of each method based on simulated and biological data under varying noise levels and p-value cutoffs. RESULTS: All methods exhibit very low false positive rates and identify a large fraction of the differentially expressed genes in simulated data sets with noise level similar to that of actual data. Overall, the rank sum test appears most conservative, which may be advantageous when the computationally identified genes need to be tested biologically. However, if a more inclusive list of markers is desired, a higher p-value cutoff or the nonparametric t-test may be appropriate. When applied to data from lung tumor and lymphoma data sets, the methods identify biologically relevant differentially expressed genes that allow clear separation of groups in question. Thus the methods described and evaluated here provide a convenient and robust way to identify differentially expressed genes for further biological and clinical analysis.

Carcinoma, Squamous Cell↗
PubMed2005

Differential expression of manganese superoxide dismutase sequence variants in near isogenic lines of wheat during cold acclimation.

Numerous sequence variants of wheat (Triticum aestivum L.) manganese superoxide dismutase (MnSOD) genes have been found. Quantitative real-time PCR was used to measure the expression levels of three MnSOD genes distinguished by a variable amino acid, and three genes distinguished by sequence variation in the 3' untranslated region (3' UTR), in wheat plants grown at 20 degrees C and cold-acclimated for 1-4 weeks at 2 degrees C. The amino acid variants did not differ significantly in expression levels, however, differential expression of genes differing in the 3' UTR was observed. Diploid wheat-related species also carried sequence variants of MnSOD, with differing levels of expression, suggesting diversification of the MnSOD gene family occurred prior to the polyploidization events of hexaploid wheat.

3' Untranslated Regions↗
PubMed2003

Identification of differentially expressed genes in shrimp (Penaeus stylirostris) infected with White spot syndrome virus by cDNA microarrays.

White spot syndrome virus (WSSV) is currently the most important viral pathogen infecting penaeid shrimp worldwide. Although considerable progress has been made in characterizing the WSSV genome and developing detection methods, information pertaining to host genes involved in WSSV pathogenesis is limited. We examined the potential of cDNA microarray analysis to study gene expression in WSSV-infected shrimp. Shrimp cDNAs were printed as low-density arrays on glass slides and were hybridized with Cy3/Cy5 labeled probes derived from RNA isolated from healthy and WSSV-infected shrimp. Genes that code for proteins that are relevant to crustacean immunity, structural proteins, as well as proteins of unknown function were among those whose mRNA expression was altered upon WSSV infection. To validate the microarray data, the temporal expression of three differentially expressed genes, an immune gene (C-type lectin-1), a structural gene (40S ribosomal protein), and a gene involved in lipid metabolism (fatty acid binding protein) was measured in healthy and WSSV-infected shrimp by real-time RT-PCR. The data suggest that WSSV infection alters the expression of a wide array of cellular genes, and provides a framework for further studies aimed at identifying genes whose function may provide insight into the mechanism of WSSV infection in shrimp.

Amino Acid Sequence↗
PubMed2005

Identification of differentially expressed cDNA transcripts from a rat odontoblast cell line.

Odontoblasts and osteoblasts are two among the myriads of cell types present in the craniofacial complex. Both have a common ectomesenchymal origin and secrete macromolecules that are necessary for the formation of dentin and alveolar bone via matrix-mediated mechanisms. The mineralized matrices of bone and dentin differ in morphology and function but several mineral associated proteins, formerly thought to be tissue specific, have been found to be common in both tissues. To decipher the complex molecular mechanisms involved in mineralized dentin formation, the suppressive subtraction hybridization (SSH) approach has been used to identify the genes expressed by polarized odontoblasts. Employing SSH, 187 cDNA clones were identified from the subtracted cDNA library. Many of these genes have not been previously reported to be expressed by terminally differentiated odontoblasts. Genes were classified into seven groups based on the predicted function of the encoded proteins: extracellular matrix; cytoskeletal components, molecules involved in adhesion and cell-cell interaction; metabolic enzymes, transporters, ion channels; protein processing, protein transport and protein folding molecules; nuclear proteins (transcription factors, DNA processing enzymes); signaling molecules and genes of yet unknown function. Northern blot and in situ hybridization analysis performed for five putative novel genes and one new isoform of amelogenin revealed differential expression levels in the osteoblasts, ameloblasts and the odontoblasts of the developing rat molars. Some of the known genes isolated from this enriched pool were the cleavage products of dentin sialophosphoprotein (DSPP) namely, phosphophoryn (PP) and dentin sialoprotein (DSP). Interestingly amelogenin, ameloblastin and enamelin were also expressed in the odontoblasts during dentin formation.

Animals↗