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Voss, Joachim G., Raghavan Raju, Carolea Logun, Robert L. Danner, Peter J. Munson, Zoila Rangel, and Marinos C. Dalakas. "A Focused Microarray to Study Human Mitochondrial and Nuclear Gene Expression." Biological Research For Nursing 9, no. 4 (April 2008): 272–79. http://dx.doi.org/10.1177/1099800408315160.
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A focused microarray (huMITOchip) was developed to study alterations of human mitochondrial and nuclear gene expression in health and disease. The huMITOchip contains 4,774 probe sets identical to the Affymetrix U 133 plus 2.0 chip covering genes affecting mitochondrial, lipid, cytokine, apoptosis, and muscle function transcripts. Unlike other gene chips, the huMITOchip has 51 probe sets that interrogate 37 genes of the mitochondrial genome. The human mitochondrial gene chip was validated against the Affymetrix U133 plus 2.0 array using an in vitro system of CCL136 muscle cell line stimulated with or without interferon gamma (IFN-γ). The 37 genes from the mtDNA demonstrated absolute gene expression levels ranging from 0.1 to 3,182. The comparison of the two gene chips yielded an excellent Pearson's correlation coefficient ( r = 0.98). At least 17 probe sets were differentially expressed in response to IFN-γ on both chips, with a high degree of concordance. This is the first report on the development of a focused oligonucleotide microarray containing genes of the mitochondrial genome.
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Memon, Farhat Naureen, Graham J. G. Upton, and Andrew P. Harrison. "A Comparative Study of the Impact of G-Stack Probes on Various Affymetrix GeneChips of Mammalia." Journal of Nucleic Acids 2010 (2010): 1–6. http://dx.doi.org/10.4061/2010/489736.
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We have previously discovered that probes containing runs of four or more contiguous guanines are not reliable for measuring gene expression in the Human HG_U133A Affymetrix GeneChip data. These probes are not correlated with other members of their probe set, but they are correlated with each other. We now extend our analysis to different3′GeneChip designs of mouse, rat, and human. We find that, in all these chip designs, the G-stack probes (probes with a run of exactly four consecutive guanines) are correlated highly with each other, indicating that such probes are not reliable measures of gene expression in mammalian studies. Furthermore, there is no specific position of G-stack where the correlation is highest in all the chips. We also find that the latest designs of rat and mouse chips have significantly fewer G-stack probes compared to their predecessors, whereas there has not been a similar reduction in G-stack density across the changes in human chips. Moreover, we find significant changes in RMA values (after removing G-stack probes) as the number of G-stack probes increases.
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Lauren, P. D. "Algorithm to model gene expression on affymetrix chips without the use of MM cells." IEEE Transactions on Nanobioscience 2, no. 3 (September 2003): 163–70. http://dx.doi.org/10.1109/tnb.2003.817020.
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MARTIGNETTI, LOREDANA, ANDREI ZINOVYEV, and EMMANUEL BARILLOT. "IDENTIFICATION OF SHORTENED 3′ UNTRANSLATED REGIONS FROM EXPRESSION ARRAYS." Journal of Bioinformatics and Computational Biology 10, no. 02 (April 2012): 1241001. http://dx.doi.org/10.1142/s0219720012410016.
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Cancer cells have been recently shown to express high level of short 3′UTR isoforms that can escape miRNA-mediated regulation. We present here a computational procedure for systematically identifying shortened 3′UTRs by Affymetrix 3′ microarrays. The advantage of this technology compared to more recent and promising ones such as exon arrays and RNA-Seq is that, giving the relatively small cost, already existing datasets in public databases include a considerably higher number of experiments. Moreover, the design of Affymetrix Gene Chips is well-suited for 3′UTR analysis of a large number of genes. Initially, Affymetrix individual probes are regrouped into customized probesets mapping specifically the CDS or the 3′UTR of the transcript, according to RefSeq annotation. Then, candidate 3′UTR shortening events are identified by statistical differential expression analysis of customized probesets in different biological conditions. The procedure has been applied to expression data from two ovarian adenocarcinoma datasets. Selected gene sets are significantly enriched for annotated splice variant genes as well as genes involved in estrogen dependent cancer mechanisms, confirming the validity of the proposed procedure.
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Fu, Li M., and Casey S. Fu-Liu. "The gene expression data of Mycobacterium tuberculosis based on Affymetrix gene chips provide insight into regulatory and hypothetical genes." BMC Microbiology 7, no. 1 (2007): 37. http://dx.doi.org/10.1186/1471-2180-7-37.
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Boes, Tanja, Elisabeth Kruse, Johannes Hüsing, and Karl-Heinz Jöckel. "P2.64: Effects of the probeset expression measure computation of gene expression data derived from Affymetrix-Chips." Biometrical Journal 46, S1 (March 2004): 153. http://dx.doi.org/10.1002/bimj.200490061.
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Dumur, Catherine I., Suhail Nasim, Al M. Best, Kellie J. Archer, Amy C. Ladd, Valeria R. Mas, David S. Wilkinson, Carleton T. Garrett, and Andrea Ferreira-Gonzalez. "Evaluation of Quality-Control Criteria for Microarray Gene Expression Analysis." Clinical Chemistry 50, no. 11 (November 1, 2004): 1994–2002. http://dx.doi.org/10.1373/clinchem.2004.033225.
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Abstract Background: Development of quality-control criteria to ensure reproducibility of microarray results for potential clinical application is still in its infancy. Methods: In the present studies we developed quality-control criteria and evaluated their effect in microarray data analysis using total RNA from cell lines, frozen tumors, and a commercially available reference RNA. Quality-control criteria such as A260/A280 ratios, percentage of rRNA, and median size of cDNA and cRNA synthesis products were evaluated for robustness in microarray analysis. Furthermore, precision studies using a reference material were performed on the Affymetrix® HG-U133A high-density oligonucleotide microarrays. The same reference RNA sample was examined in 16 different chips run on 2 different days in the four different modules of the Affymetrix fluidics workstation. Fresh and frozen fragmented cRNAs were also compared. An ANOVA model was fit to identify the main sources of variation. Results: Good-quality samples showed >30% rRNA in the electropherograms and cDNA and cRNA synthesis products with median sizes of 2.0 and 3.0 kb, respectively. Precision studies showed that the main source of variation was the day-to-day variability, minimally affecting hybridization exogenous control genes. Altogether, the results showed that the Affymetrix Genechip® system is highly reproducible when RNA that meet the quality-control criteria are used (overall P >0.01). Conclusions: These results confirm the need to establish defined quality-control criteria for sample quality to distinguish between analytical and biological variability.
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Misson, J., K. G. Raghothama, A. Jain, J. Jouhet, M. A. Block, R. Bligny, P. Ortet, et al. "A genome-wide transcriptional analysis using Arabidopsis thaliana Affymetrix gene chips determined plant responses to phosphate deprivation." Proceedings of the National Academy of Sciences 102, no. 33 (August 5, 2005): 11934–39. http://dx.doi.org/10.1073/pnas.0505266102.
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Jung, Sin-Ho, and Insuk Sohn. "Statistical Issues in the Design and Analysis of nCounter Projects." Cancer Informatics 13s7 (January 2014): CIN.S16343. http://dx.doi.org/10.4137/cin.s16343.
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Numerous statistical methods have been published for designing and analyzing microarray projects. Traditional genome-wide microarray platforms (such as Affymetrix, Illumina, and DASL) measure the expression level of tens of thousands genes. Since the sets of genes included in these array chips are selected by the manufacturers, the number of genes associated with a specific disease outcome is limited and a large portion of the genes are not associated. nCounter is a new technology by NanoString to measure the expression of a selected number (up to 800) of genes. The list of genes for nCounter chips can be selected by customers. Due to the limited number of genes and the price increase in the number of selected genes, the genes for nCounter chips are carefully selected among those discovered from previous studies, usually using traditional high-throughput platforms, and only a small number of definitely unassociated genes, called control genes, are included to standardize the overall expression level across different chips. Furthermore, nCounter chips measure the expression level of each gene using a counting observation while the traditional high-throughput platforms produce continuous observations. Due to these differences, some statistical methods developed for the design and analysis of high-throughput projects may need modification or may be inappropriate for nCounter projects. In this paper, we discuss statistical methods that can be used for designing and analyzing nCounter projects.
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Haerizadeh, Farzad, Mohan B. Singh, and Prem L. Bhalla. "Transcriptome profiling of soybean root tips." Functional Plant Biology 38, no. 6 (2011): 451. http://dx.doi.org/10.1071/fp10230.
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Soybean (Glycine max L.), a major legume crop, is important to human nutrition and is a source of animal feed. Similar to many legumes, a key feature of the soybean is its symbiotic association with soil bacteria that fix atmospheric nitrogen. However, knowledge of the gene expression of its root system, particularly the root meristematic region, is limited. Here, we have addressed this by investigating the gene expression profile of the soybean root tip, using soybean Affymetrix chips containing 37 500 probe sets (Affymetrix Inc.) and have compared this expression profile with that of the nonmeristematic tissue. We identified a total of 5012 upregulated and 4136 downregulated genes in the soybean root tip. Among the upregulated genes, 559 showed strong preferential expression in the root tip, indicating that they are likely to be associated with root apical meristem specificity and root tip function. Genes involved in membrane transport, defence signalling and metabolism were upregulated in the soybean root tip. Further, our data provide a resource of novel target genes for further studies involving root development and biology, and will possibly have a positive impact on future crop breeding.
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Liu, Lixin, Jiang Zhu, Peter S. A. Glass, Peter R. Brink, Ira J. Rampil, and Mario J. Rebecchi. "Age-associated Changes in Cardiac Gene Expression after Preconditioning." Anesthesiology 111, no. 5 (November 1, 2009): 1052–64. http://dx.doi.org/10.1097/aln.0b013e3181bbcb2a.
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Background Cardiac protection afforded by ischemic preconditioning (IPC) and anesthetic preconditioning (APC) are significantly reduced in the senescent myocardium. The authors hypothesized that age would differentially modulate gene expression induced by IPC and APC in vivo. Methods Affymetrix RAT EXON ST 1.0 gene chips (Affymetrix, Santa Clara, CA) were used to explore the transcriptional response to IPC and APC in Fisher 344 male rats (young, 3-5 months, and old, 20-24 months, respectively). Both cohorts, young and old, were divided into three groups: (1) sham control, (2) IPC, and (3) APC. After a total of 90 min, the heart was removed, and the total RNA and protein were extracted. Results Thirty-one transcripts were increased in the young animals subjected to IPC, particularly transcriptional regulators (Atf3, Egr-1, Btg2, Egr2), cytokines (interleukin 6, CSF1, Myd88), chemokines (Cxcl10, Ccl2, Ccl7), regulators of growth and inflammation (Reg3g, Hamp), remodeling and cell adhesion migration (Cyr61, Tfpi2, Timp1), regulators of apoptosis/cell death (Birc3, Arntl, Hamp, Phlda1), and cell cycle control/DNA repairs (Rrad, Gadd45b, Gadd45g). In contrast, only one transcript increased (Atf3) in the old animals subjected to IPC. No changes in gene expression were found in the young or the old animals subjected to APC. Conclusions Early-phase IPC and APC induced different genomic responses. The absence of detectable changes associated with early-phase APC suggests a posttranscriptional or posttranslational mechanism. The absence of a genomic response in the senescent myocardium (except for IPC-induced Atf3) could underlie the failure of IPC to provide any cardiac protective benefit to older animals.
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Van Laere, S. J., I. Van der Auwera, G. G. Van den Eynden, X. Trinh, P. Van Hummelen, P. van Dam, E. A. Van Marck, P. B. Vermeulen, and L. Y. Dirix. "Confirmation of the distinct molecular phenotype of inflammatory breast cancer compared to non-inflammatory breast cancer using Affymetrix-based genome-wide gene expression analysis." Journal of Clinical Oncology 25, no. 18_suppl (June 20, 2007): 21055. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.21055.
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21055 Background: We have shown with cDNA microarrays that inflammatory breast cancer (IBC) and non-IBC are distinct biological entities. The purpose of this study was to confirm our previous results using Affymetrix chips. Methods: RNA was extracted from 19 IBC samples and 42 non-stage matched non-IBC samples. RNA was hybridized onto Affymetrix HG U133 Plus 2.0 chips. Gene expression data were normalized using GCRMA and genes with a gene expression of at least 250 in 50% of the cases were filtered in. Hierarchical clustering and principle component analysis was executed. Identification of the different cell-of-origin subtypes in our expression data set was done using the intrinsic gene list. A NFkB signature, a MAPK signature and our own IBC signature were tested by clustering analysis. Results: Clustering using 11341 genes resulted in the identification of two clusters: one containing 14/19 IBC samples and a second containing 32/42 non-IBC (Pearson χ2; p<0.0001). Principle component analysis separated IBC from non-IBC samples along the first principle component. Interestingly, IBC samples more closely resemble T1 - T2 tumours than T3 - T4 tumours. Application of the intrinsic gene set to our IBC/non-IBC data set resulted in the classification of 14/19 IBC samples as basal-like or ErbB2-overexpressing tumours compared to only 4/42 non-IBC tumours (Pearson χ2; p<0.0001). Our own IBC signature was confronted with the new data set and performed well in separating IBC specimens form non-IBC specimens. Clustering identified three clusters from which one cluster contained 18 samples, including 12 IBC specimens (p<0.0001). Using the NFkB and MAPK signatures, similar results were obtained. Conclusions: These results confirm our findings that IBC is a distinct biologic phenotype, characterized by activation of NFkB, possibly through activation of MAPK's. IBC tumours more often demonstrate characteristics from basal-like and ErbB2-overexpressing breast tumours. The fact that IBC tumours are rapidly developing tumours instead of longstanding tumourigenic processes might explain the close resemblance of the IBC gene expression profile to the gene expression profile of T1 and T2 tumours. No significant financial relationships to disclose.
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Menyhárt, Otília, Ádám Nagy, and Balázs Győrffy. "Determining consistent prognostic biomarkers of overall survival and vascular invasion in hepatocellular carcinoma." Royal Society Open Science 5, no. 12 (December 2018): 181006. http://dx.doi.org/10.1098/rsos.181006.
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Background: Potential prognostic biomarker candidates for hepatocellular carcinoma (HCC) are abundant, but their generalizability is unexplored. We cross-validated markers of overall survival (OS) and vascular invasion in independent datasets. Methods: The literature search yielded 318 genes related to survival and 52 related to vascular invasion. Validation was performed in three datasets (RNA-seq, n = 371; Affymetrix arrays, n = 91; Illumina gene chips, n = 135) by uni- and multivariate Cox regression and Mann–Whitney U -test, separately for Asian and Caucasian patients. Results: One hundred and eighty biomarkers remained significant in Asian and 128 in Caucasian subjects at p < 0.05. After multiple testing correction BIRC5 ( p = 1.9 × 10 −10 ), CDC20 ( p = 2.5 × 10 −9 ) and PLK1 ( p = 3 × 10 −9 ) endured as best performing genes in Asian patients; however, none remained significant in the Caucasian cohort. In a multivariate analysis, significance was reached by stage ( p = 0.0018) and expression of CENPH ( p = 0.0038) and CDK4 ( p = 0.038). KIF18A was the only gene predicting vascular invasion in the Affymetrix and Illumina cohorts ( p = 0.003 and p = 0.025, respectively). Conclusion: Overall, about half of biomarker candidates failed to retain prognostic value and none were better than stage predicting OS. Impact: Our results help to eliminate biomarkers with limited capability to predict OS and/or vascular invasion.
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Kelly, Danny J., and Sujoy Ghosh. "RNA Profiling for Biomarker Discovery: Practical Considerations for Limiting Sample Sizes." Disease Markers 21, no. 1 (2005): 43–48. http://dx.doi.org/10.1155/2005/357089.
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We have compared microarray data generated on Affymetrix™chips from standard (8 micrograms) or low (100 nanograms) amounts of total RNA. We evaluated the gene signals and gene fold-change estimates obtained from the two methods and validated a subset of the results by real time, polymerase chain reaction assays. The correlation of low RNA derived gene signals to gene signals obtained from standard RNA was poor for less to moderately abundant genes. Genes with high abundance showed better correlation in signals between the two methods. The signal correlation between the low RNA and standard RNA methods was improved by including a reference sample in the microarray analysis. In contrast, the fold-change estimates for genes were better correlated between the two methods regardless of the magnitude of gene signals. A reference sample based method is suggested for studies that would end up comparing gene signal data from a combination of low and standard RNA templates; no such referencing appears to be necessary when comparing fold-changes of gene expression between standard and low template reactions.
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Sergeev, Pavel, Rafaela da Silva, Eliana Lucchinetti, Kathrin Zaugg, Thomas Pasch, Marcus C. Schaub, and Michael Zaugg. "Trigger-dependent Gene Expression Profiles in Cardiac Preconditioning." Anesthesiology 100, no. 3 (March 1, 2004): 474–88. http://dx.doi.org/10.1097/00000542-200403000-00005.
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Background DNA chips facilitate genomic-wide exploration of gene expression. The authors hypothesized that ischemic (IPC) and anesthetic preconditioning (APC) would differentially modulate gene expression in hearts. Methods Affymetrix rat U34A gene chips were used to explore the transcriptional response to IPC and APC, sustained ischemia (110 min) without reperfusion, and time-matched perfusion in isolated rat hearts. IPC was induced by three cycles of 5 min of ischemia, and APC was induced by 1.5 minimum alveolar concentration isoflurane (110 min). For each heart, a separate chip was used for hybridization. Data were analyzed for significant > or = 2.0-fold changes in gene expression. Microarray results were confirmed by quantitative real-time reverse-transcription polymerase chain reaction. Results Of the 8,799 genes represented on U34A, 217 transcripts in the APC group, 234 in the IPC group, and 29 in the ischemia group displayed significant > or = 2.0-fold up-regulation in messenger RNA levels, and 185 transcripts in the APC group, 55 in the IPC group, and 49 in the ischemia group displayed significant > or = 2.0-fold down-regulation. Many of these transcripts were unknown genes. A high number of commonly regulated genes were found in IPC and APC (39 up-regulated, 17 down-regulated). Genes commonly regulated included those associated with cell defense (heat shock protein 10, aldose reductase, Bcl-xS). Conversely, a pool of protective and antiprotective genes was differentially regulated in APC versus IPC (heat shock protein 27/70, programmed cell death 8), suggesting trigger-dependent transcriptome variability. Conclusions The novel microarray technology provides evidence for distinct cardioprotective phenotypes in IPC and APC. The observed transcriptional changes raise the possibility of a second window of protection by volatile anesthetics. The authors' molecular portraits are the first global genomic comparison between IPC and APC.
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Lankford, Amy R., Anne M. Byford, Kevin J. Ashton, Brent A. French, Jae K. Lee, John P. Headrick, and G. Paul Matherne. "Gene expression profile of mouse myocardium with transgenic overexpression of A1adenosine receptors." Physiological Genomics 11, no. 2 (October 29, 2002): 81–89. http://dx.doi.org/10.1152/physiolgenomics.00008.2002.
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Transgenic mice with cardiac-specific overexpression of adenosine A1receptors (A1AR) have demonstrated metabolic and functional tolerance to myocardial ischemia. We utilized cDNA microarrays to test the hypothesis that the cardioprotective mechanism(s) of A1overexpression involves altered gene expression. Total RNA extracted from the left ventricles from A1transgenic ( n = 4) and wild-type ( n = 6) mice was hybridized to Affymetrix mgU74A chips. Comparison of RNA expression levels in transgenic to wild-type myocardium revealed ∼636 known genes with expression significantly altered by greater than 25%. We observed increased expressions of genes including NADH dehydrogenase, the GLUT4 glucose transporter, Na-K-ATPase, sarcolemmal KATPchannels, and Bcl-xl in A1AR-overexpressing hearts. We also observed decreased expression of pro-apoptotic genes including a 50% reduction in message level of caspase-8. Protein expression of GLUT4 and caspase-8 was also altered comparable to the differences in gene expression. These data illustrate genes with chronically altered patterns of expression in A1transgenic mouse myocardium that may be related to adenosine receptor overexpression-mediated cardioprotection.
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Reme, Thierry X., Dirk Hose, John De Vos, Hartmut Goldschmidt, Friedrich Cremer, and Bernard Klein. "A New Method for Predictor Calculation Based on Signed-Rank Call Algorithms. Application to Microarray Gene Expression Analysis of Human Multiple Myeloma." Blood 104, no. 11 (November 16, 2004): 4876. http://dx.doi.org/10.1182/blood.v104.11.4876.4876.
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Abstract The simultaneous quantification of thousands of genes in gene expression profiling (GEP) on DNA chips is part of the whole-genome sequencing revolution. Affymetrix(R) chip technology provides both a quantitative fluorescence signal and a decision of absent or present gene expression (absent or present call) based on signed-rank algorithms applied to several hybridization repeats of each gene spread on a single chip. To avoid an empirical normalization between chips of the same experiment, we developed an analysis of GEP based on Affymetrix present or absent calls. Bone marrow aspirates from newly-diagnosed multiple myeloma (MM) patients were purified with CD138 automated magnetic cell sorting. Amplified RNA was run on U133A+B Affymetrix DNA microarrays for a first set of 65 patients, or U133Plus2 for a second cohort of 40 patients. Scan files were transferred to an Oracle(R) data base and analyzed with web-oriented scripts for both unsupervised and supervised non-parametric analysis on either the fluorescence signal or the Affymetrix call. To build a multiclass call-based predictor, the observed distribution of present call of each probeset was first compared between predetermined sample groups using a chi2 test and probesets were kept only if above a threshold compatible with further analysis and calculation time (usually 100 to 1000 genes). The power of a probeset list to classify the different groups (number of presence/number of probesets) was then evaluated for each sample of a group and compared to each sample of all other groups by calculating the reduced deviation (RD) in paired comparisons and evaluating the overall number of non significant comparisons (NS) with a chosen precision, the sum of the reduced deviations divided by the square root of the probeset number (f, independent of the list size), and the smallest RD (RDmin). The minimum predictive probeset list was obtained by deleting each probeset one after the other, and computing NS, f and RDmin from the remaining probeset list. If either NS is reduced, or both NS unchanged and f increased, or together NS and f unchanged and RDmin either increased or higher than precision, the probeset is left out and the process run again on the shortened list until no more leave-outs are possible. This method was successfully validated by determining a 22-gene sex predictor with the 65 patient series that made it possible to classify gender with no error in the 40 patient validation group. Partial loss of chromosome Y was confirmed in 3 male MM patients by short tandem repeat analysis. Significant predictors could not be generated with randomly selected patient groups. Validation was also successful with P <.001 in predicting the immunoglobulin light chain of the validation group after educating with the training group (lambda to kappa-type ratio between 1/4 and 1/3). Classification of the training set according to Salmon-Durie staging made it possible to generate a 97-gene predictor with a validation error of P <.01. This normalization-free method looks particularly promising for further applications like diagnostic classification (MGUS), prognostic grouping and prediction of response to treatment. In addition, it can be used as a powerful tool to mine generated or published data on all cancer types.
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Hartmann, O. "Quality Control for Microarray Experiments." Methods of Information in Medicine 44, no. 03 (2005): 408–13. http://dx.doi.org/10.1055/s-0038-1633985.
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Summary Objectives: In this paper we give an overview of post-hybridization quality control methods for gene expression chips, including methods for the gene/spot level, the hybridization/chip level and the process level. We present quality control methods that can be applied after hybridization and image analysis, i.e. that use data from the chip experiment itself. Wet lab quality control steps, which should be applied before the probe is measured on a chip, are not discussed. This review is aimed towards statisticians and data analysts. Methods: We give examples of some of the quality control measures available for spotted cDNA and Affymetrix GeneChips®, the most common chip types. As quality control measures are technology and design-dependent, we will stress on methods that have the potential to be applied platform-independently. Results: Quality control should identify poor quality chips or hybridizations, as well as faulty measurements for individual genes/spots. Additionally, high throughput laboratories processing several tens or hundreds of microarrays per week have the need for an appropriate process control to be able to identify changes in the production process as early as possible. Conclusion: Microarrays have become a standard research tool for biologists and medical researchers. As a consequence, there is a great need for standardized quality control, as false findings due to problem in data quality can lead to a substantial loss of resources.
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Cortez, Karoll J., Caron A. Lyman, Shyam Kottilil, Hee Sup Kim, Emmanuel Roilides, Jun Yang, Brandie Fullmer, Richard Lempicki, and Thomas J. Walsh. "Functional Genomics of Innate Host Defense Molecules in Normal Human Monocytes in Response to Aspergillus fumigatus." Infection and Immunity 74, no. 4 (April 2006): 2353–65. http://dx.doi.org/10.1128/iai.74.4.2353-2365.2006.
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ABSTRACT Aspergillus fumigatus induces the release of innate immune-related molecules from phagocytic cells early in the course of infection. Little is known, however, about the complex expression profiles of the multiple genes involved in this response. We therefore investigated the kinetics of early gene expression in human monocytes (HMCs) infected with conidia of A. fumigatus using DNA microarray analysis. Total RNA from HMCs at 0, 2, 4, and 6 h was extracted, linearly amplified, hybridized onto Affymetrix HG133 Plus 2.0 gene chips, and analyzed with an Affymetrix scanner. Changes in gene expression were calculated as a ratio of those expressed by infected versus control HMCs. Aspergillus fumigatus induced differential regulation of expression in 1,827 genes (P < 0.05). Genes encoding cytokines and chemokines involved in host defense against A. fumigatus, including interleukin-1β (IL-1β), IL-8, CXCL2, CCL4, CCL3, and CCL20, as well as the opsonin long pentraxin 3, were up-regulated during the first 2 to 6 h, coinciding with an increase in phagocytosis. Simultaneously, genes encoding CD14, ficolin1, and MARCO were down-regulated, and genes encoding IL-10 and matrix metalloproteinase 1 were up-regulated. Up-regulation of the genes encoding heat shock proteins 40 and 110 and connexins 26 and 30 may point to novel molecules whose role in the pathogenesis of aspergillosis has not been previously reported. Verification of the transcriptional profiling was obtained for selected genes by reverse transcription-PCR and enzyme immunoassay. Thus, A. fumigatus conidia induced a coordinated expression of genes important in host defense and immunomodulation.
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Johnson, R. D., S. Borchert, M. J. Christensen, L. J. Johnson, A. Koulman, M. J. Van Gils, and G. T. Bryan. "A gene identified from Neotyphodium lolii is expressed only in planta and regulates the biosynthesis of a putative oligopeptide secondary metabolite." NZGA: Research and Practice Series 13 (January 1, 2007): 485–89. http://dx.doi.org/10.33584/rps.13.2006.3132.
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Endophytes, belonging to the genus Neotyphodium, live symptomlessly within the intercellular spaces of cool-season grasses, and confer a number of biotic and abiotic advantages to their hosts. We identified a novel endophyte gene (designated Nc25) that is expressed preferentially in planta, is one of the most abundant fungal transcripts in endopyte-infected grasses and which is distributed and highly expressed in a wide range of endophyte/ grass associations. Nc25 is novel and shows no homology to sequence databases or fungal genome initiatives. Characterisation indicates that it encodes a small secreted protein. Re-introduction of a Nc25 deletion strain into perennial ryegrass showed no visible effect on the symbiosis but an unknown oligopeptide, detected only in infected plants, was eliminated. Surprisingly, the oligopeptide is unrelated to the predicted peptide product of Nc25. We hypothesise that Nc25 may regulate the oligopeptide biosynthetic pathway and are investigating this using Affymetrix gene chips to determine how Nc25 affects global gene expression. In addition we are interested in the biological function of this secondary metabolite during symbiosis and in particular whether it has bioactivity that may confer abiotic or biotic advantages to the host plant. Keywords: Neotyphodium, in planta expressed gene, oligopeptide, EGFP
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DAI, HENG, BIN TIAN, WEI D. ZHAO, ALBERT LEUNG, SIMON R. SMITH, JACKSON S. WAN, and XIANG YAO. "DYNAMIC INTEGRATION OF GENE ANNOTATION AND ITS APPLICATION TO MICROARRAY ANALYSIS." Journal of Bioinformatics and Computational Biology 01, no. 04 (January 2004): 627–45. http://dx.doi.org/10.1142/s0219720004000387.
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Comprehensive and structured annotations for all genes on a microarray chip are essential for the interpretation of its expression data. Currently, most chip gene annotations are one-line free text descriptions that are often partial, outdated and unsuitable for large-scale data analysis. Therefore the interpretation of microarray gene expression clusters is often limited. Although researchers can manually navigate a collection of databases for better annotations, it is only practical for limited number of genes. Existing meta-databases fail to provide comprehensive categorized annotations for hundreds of genes simultaneously. We have developed an automatic system to address this issue. GeneView system monitors various data sources, extracts gene information from a source whenever it is updated, comprehensively matches genes, and integrates them into a central database by categories, such as pathway, genetic mapping, phenotype, expression profile, domain structure, protein interaction, disease association, and references. The system consists of four major components: (1) relational database; (2) data processing; (3) user curation; (4) data query. We evaluated it by analyzing genes on cDNA and Affymetrix Oligo chips. In both cases, the system provided more accurate and comprehensive information than those provided by the vendors or the chip users, and helped identify new common functions among genes in the same expression clusters.
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Carter, F., T. Fair, S. Park, M. Wade, A. C. O. Evans, and P. Lonergan. "263 GENE EXPRESSION PROFILING OF IMMATURE AND IN VITRO-MATURED BOVINE OOCYTES USING AFFYMETRIX GENECHIP TECHNOLOGY." Reproduction, Fertility and Development 19, no. 1 (2007): 248. http://dx.doi.org/10.1071/rdv19n1ab263.
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Previous studies by our group have demonstrated that oocyte maturation is a crucial event in the determination of subsequent developmental competence. The objective of the current study was to characterize changes in gene expression profiles of bovine oocytes during meiotic maturation. To this end, 5 replicate pools of 200 bovine cumulus–oocyte complexes (COCs)were collected from the ovaries of slaughtered heifers. Upon recovery, 100 COCs from each replicatewere immediately denuded, and the oocytes were snap frozen in liquid nitrogen. The remaining 100 COCs were matured in vitro in TCM-199 supplemented with 10% (v/v) fetal calf serum and 10 ngmL-1 epidermal growth factor for 24 h at 39�C under an atmosphere of 5% CO2 in air with maximum humidity. Following maturation, the remaining COCs were denuded and snap frozen. Total RNA was isolated (mean total RNA content 106.08�38.87 ng per 100 oocytes) and subjected to 2 rounds of amplification incorporating biotin-labeled nucleotides during the second in vitro transcription reaction (mean total RNA content 155.15�51.14 �g per 100 oocytes post-amplification). The resulting labeled antisense RNA was hybridized to a GeneChip Bovine Genome Arrays (Affymetrix, Inc., Santa Clara, CA, USA) (10 chips, 5 replicates each of immature and mature oocytes, n=100 oocytes/chip). Expression data were analysed using Genespring software (Agilent Technologies, Palo Alto, CA, USA), and data were normalized to the median. Overall, 54.9�1.3% and 53.3�3.3% of the 24 178 probe sets representing 23 000 transcripts spotted on the arrays were expressed in immature and in vitro-matured oocytes, respectively. Across the 5 array comparisons, 52 genes were consistently exclusively present in immature oocytes, whereas 16 genes were exclusively present in mature oocytes. A further 821 genes were found to be differentially expressed (≥2-fold) between the 2 groups (P <0.05), of which 209 were up-regulated and 612 were down-regulated in the in vitro-matured oocytes compared with their immature counterparts. The differentially expressed transcripts were classified according to their gene ontology (http://benzer.ubic.ca/ermineJ). The existing Affymetrix annotation was updated by blasting the sequences against bovine, human, and murine databases (≥90% homology; increasing molecular function annotation from 14% to 42%). In terms of olecular function, the majority of these genes were associated with protein or nucleic acid binding (>42%), catalytic activity (24%), signal transduction (7%), transporter activity (5%), and structural molecule activity (5%). In conclusion, we have stablished the molecular transcriptome blueprint of immature and in vitro-matured bovine oocytes. Through comparisons with in vivo-matured oocytes, this resource will be invaluable in determining genes that are involved in controlling the developmental competence of oocytes. This research was funded by the Science Foundation Ireland (02/IN1/B78).
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Abdelkefi, Abderrahman, John de Vos, Said Assou, Tarek Ben Othman, Jean-Francois Rossi, Saloua Ladeb, Ines Safra, et al. "Gene Expression Profiling of Myeloma Cells To Predict Response to Primary Therapy with Thalidomide-Dexamethasone for Newly Diagnosed Multilple Myeloma." Blood 110, no. 11 (November 16, 2007): 1494. http://dx.doi.org/10.1182/blood.v110.11.1494.1494.
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Abstract Background: Thalidomide which represents an effective treatment strategy for relapsed/refractory multiple myeloma, actually represents a standard of care also for newly diagnosed multiple myeloma patients. Methods: In the present study, we adopted a gene expression profiling (GEP) strategy in an attempt to predict response (> 50% reduction in serum M protein) to primary therapy with thalidomide-dexamethasone for newly diagnosed multiple myeloma. Plasma cells (CD138+) were purified from bone marrow aspirates from 17 patients at diagnosis, before initiation of treatment with thalidomide-dexamethasone. GEP was performed using the Affymetrix U133 Plus_2 microarray platform. The Affymetrix output (CEL files) was imported into Genespring 7.3 (Agilent technologies) microarray analysis software, where data files were normalized across chips using GCRMA and to the 50th percentile, followed by per gene normalization to median. Criteria of response were those established by Bladè et al. Results: After sufficient follow-up, responders (n=9) and nonresponders (n=8) were identified, and gene expression differences in baselines samples were examined. Of the 11000 genes surveyed, Wilcoxon rank sum test identified 149 genes that distinguished response from non response. A multivariate step-wise discriminant analysis (MSDA) revealed that 14 of the 149 genes could be used in a response predictor model (see table). Of interest, the gene list encompasses WXSC1, known to be involved in the chromosomal translocation t(4;14) (p16.3;q32.3) in multiple myeloma. Conclusion: These results could be the first step to adopt microfluidic cards, in an attempt to select at diagnosis patients who will respond favourably to a particular treatment strategy. List of 14 genes able to predict response to primary therapy with thalidomide-dexamethasone for newly diagnosed multiple myeloma. Gene ID Gene Name Chromosomal location 212771_at C10orf38 10p13 229874_x_at LOC400741 1p36.13 219690_at U2AF1L4 19q13.12 202207_at ARL7 2q37.1 243819_at GNG2 14q21 203753_at TCF4 18q21.1 235400_at FCRLM1 1q23.3 211474_s_at SERPINB6 6p25 226785_at ATP11C Xq27.1 215440_s_at BEXL1 Xq22.1–q22.3 209054_s_at WXSC1 4p16.3 227168_at FLJ25967 22p12.1 213355_at ST3GAL6 3q12.1 223218_s_at NFKBIZ 3p12–q12
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Holmes, F. A., J. A. O’Shaughnessy, B. Hellerstedt, J. Pippen, S. Vukelja, D. Kocs, L. Asmar, et al. "Pharmacogenomic analysis of needle biopsies obtained before preoperative docetaxel/capecitabine/FEC (TX/FEC) chemotherapy for breast cancer." Journal of Clinical Oncology 24, no. 18_suppl (June 20, 2006): 10595. http://dx.doi.org/10.1200/jco.2006.24.18_suppl.10595.
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10595 Background: Our goal was to evaluate the feasibility of obtaining fine needle biopsies, for pharmacogenomic analysis, in community based oncology practices and develop gene expression-based predictors of pathologic complete response (pCR) to preoperative sequential docetaxel/capecitabine and 5-fluorouracil, epirubicin, cyclophosphamide chemotherapy. Methods: One hundred seventy-five patients were accrued at 29 sites in the US Oncology Research network. FNA specimens were mailed to a central laboratory (MDACC) and gene expression profiling was performed on Affymetrix U133A chips. Results: RNA extraction was started on 140 specimens, 112 of these (80%) yielded ≥1 μg total RNA, 69 were hybridized and 65 (94%) gene expression profiles have passed quality control as of abstract submission date. The analysis plan is to develop a multigene predictor of pCR from the first 80 cases and test its performance independently in the remaining cases. Conclusions: Collection of mandatory research FNA biopsies for pharmacogenomic research is feasible in community practice. Approximately 80% of biopsies yield sufficient RNA for gene expression profiling. In 20% of patients, either technical factors, which can be addressed, or tumor biology (necrotic, rapidly growing tumors) were limiting. Supported by Roche Laboratories, Inc., Nutley, NJ; Pfizer, New York, NY; and Precision Therapeutics, Pittsburgh, PA. [Table: see text]
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Almon, Richard R., Debra C. DuBois, Jin Y. Jin, and William J. Jusko. "Temporal profiling of the transcriptional basis for the development of corticosteroid-induced insulin resistance in rat muscle." Journal of Endocrinology 184, no. 1 (January 2005): 219–32. http://dx.doi.org/10.1677/joe.1.05953.
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Elevated systemic levels of glucocorticoids are causally related to peripheral insulin resistance. The pharmacological use of synthetic glucocorticoids (corticosteroids) often results in insulin resistance/type II diabetes. Skeletal muscle is responsible for close to 80% of the insulin-induced systemic disposal of glucose and is a major target for glucocorticoid-induced insulin resistance. We used Affymetrix gene chips to profile the dynamic changes in mRNA expression in rat skeletal muscle in response to a single bolus dose of the synthetic glucocorticoid methyl-prednisolone. Temporal expression profiles (analyzed on individual chips) were obtained from tissues of 48 drug-treated animals encompassing 16 time points over 72 h following drug administration along with four vehicle-treated controls. Data mining identified 653 regulated probe sets out of 8799 present on the chip. Of these 653 probe sets we identified 29, which represented 22 gene transcripts, that were associated with the development of insulin resistance. These 29 probe sets were regulated in three fundamental temporal patterns. 16 probe sets coding for 12 different genes had a profile of enhanced expression. 10 probe sets coding for eight different genes showed decreased expression and three probe sets coding for two genes showed biphasic temporal signatures. These transcripts were grouped into four general functional categories: signal transduction, transcription regulation, carbohydrate/fat metabolism, and regulation of blood flow to the muscle. The results demonstrate the polygenic nature of transcriptional changes associated with insulin resistance that can provide a temporal scaffolding for translational and post-translational data as they become available.
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Goy, Andre, John Stewart, Bedia Barkoh, Yvonne Remache, Susan Hart, Susan Neal, Ruth Katz, Nour Sneige, and Frederic Gilles. "Feasibility of Gene Expression Profiling Generated from Fine Needle Aspiration from Patients with Follicular Lymphoma and Large B-Cell Lymphoma." Blood 104, no. 11 (November 16, 2004): 4315. http://dx.doi.org/10.1182/blood.v104.11.4315.4315.
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Abstract We tested the feasibility of performing gene expression profiling using amplified RNA from Fine Needle Aspiration (FNA) obtained from lymph nodes. Twenty-four samples from patients with a diagnosis of Follicular Lymphoma (FL) or Large B-Cell Lymphoma (LBCL) were obtained after informed consent. The diagnosis were performed by two pathologists and classified into two groups (10 LBCL and 14 FL) using conventional morphology and immunophenotyping. Total RNA was extracted using RNaqueous (Ambion, Austin, TX). Total RNA (100ng /per sample) was subjected to 2 cycles of standard double-stranded cDNA synthesis using Superscript Choice System (Invitrogen, Grand Island, NY) and in vitro transcription for target amplification as per GeneChip’s Eukaryotic Small Sample Target Labeling protocol (Affymetrix, Inc, Santa Clara, CA). The biotinylated cRNA from each sample was hybridized to Affymetrix HG-U133A chips. Gene expression profiling results were first analyzed by Principal Component Analysis (PCA) using a list of 146 probe sets representing 62 genes characteristic of Acivated B-Cell (ABC) or Germinal Center (GC) signature. This analysis identified 5 LBCL cases with ABC cell signature (Fig 1). Using a list of 207 probe sets representing 113 genes involved in FL transformation (K Elenitoba-Johnson, PNAS 2003), PCA analysis identified two overlapping clusters corresponding to FL and GC-DLBCL. To further improve this classification, we generated a list of 82 genes differentially expressed between FL and GC-LBCL. Using this list of genes, PCA analysis demonstrated a clear separation between FL and GC-LBCL (Fig 2). These results demonstrate that comprehensive transcription profiles can be performed in patients with NHL using RNA obtained from FNA and that one can distinguish among FL and LBCL lymphoma genetic subtypes. Figure Figure
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Tsyganov, M. M., M. K. Ibragimova, A. M. Pevzner, and N. V. Litviakov. "LOSS OF HETEROZYGOSITY IN THE BRCA1 AND BRCA2 LOCUS IN BREAST CANCER." Siberian journal of oncology 19, no. 3 (July 6, 2020): 97–101. http://dx.doi.org/10.21294/1814-4861-2020-19-3-97-101.
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One of the factors of variability of malignant neoplasms is the loss of heterozygosity (LOH). The biological meaning of LOH, in relation to carcinogenesis, is associated with the inactivation of heterozygous loci of pathogenetically significant genes. Thus, the aim of this work was to study BRCA1/2 LOH in breast tumors.Material and Methods. The study included 122 patients with stage IIAIIIC breast cancer. DNA was isolated from 122 biopsy samples of tumor tissue using the QIAamp DNA mini Kit (Qiagen, Germany). To assess the status of LOH, microarray analysis was performed on high-density DNA chips from Affymetrix CytoScanTM HD Array. To process the results of microchipping, we used the Chromosome Analysis Suite 3.3 program (Affymetrix, USA).Results. The loss of heterozygosity in the BRCA1 gene was found to be associated with response to NAC. It was shown that in 59 patients LOH in the BRCA1gene was associated with an objective response to treatment (p=0.005). The presence of LOH in the studied genes was associated with a favorable prognosis. The 5-year non-metastatic survival rates were 75 % and 100 % in patients with LOH in the BRCA1 and BRCA2 genes, respectively (log-rank test: p=0.003 and p=0.05, respectively).Conclusion. The phenomenon of LOH in the BRCA1/2 genes was shown to be associated with response to NACT. BRCA1/2. Further studies are needed to evaluate the frequency of BRCA1/2 LOH after NAC for choosing and changing treatment tactics.
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Wang, Jingjing, Yongyan Zhao, Kecui Gu, Ping Yu, Baole Zhang, Wei Wang, Juanjuan Yang, and Yinxue Xu. "The novel porcine gene early growth response 4 (Egr4) is differentially expressed in the ovaries of Erhualian and Pietrain pigs." Reproduction, Fertility and Development 26, no. 4 (2014): 587. http://dx.doi.org/10.1071/rd12380.
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The early growth response 4 (Egr4) gene plays a critical role in human and mouse fertility. In the present study, Affymetrix microarray gene chips were used to evaluate differential gene expression in the ovaries between Erhualian and Pietrain pigs. In all, 487 and 573 transcripts were identified with significantly higher and lower expression, respectively, in Erhualian compared with Pietrain sows. The Egr4 gene, one of the differentially expressed genes, was cloned and its genomic structure was analysed. Egr4 expression is increased 120-fold in ovaries from Erhualian sows. The full-length cDNA of porcine Egr4 was obtained by in silico cloning and 5′ rapid amplification of cDNA ends. The gene consists of two exons and its predicted protein contains a Cys2His2 zinc finger structure. The porcine transcript is alternatively spliced by exon sequence deletion, producing two different mRNAs differing at the 5′ end of Exon 2. Egr4 transcripts were detected in the central nervous system, including the cerebrum, cerebellum, hypothalamus and pituitary gland, and were highly expressed in the ovary. The Egr4 gene was evaluated as a candidate gene for porcine reproductivity. To investigate the role of Egr4 in the ovary, Egr4 was knocked down using short interference (si) RNA in porcine granulosa cells. Knockdown of Egr4 using siRNA effectively inhibited Egr4 mRNA and protein expression and knockdown significantly affected the expression of Bax, P450arom, P450scc, Egr1, Egr2, and Egr3. In conclusion, these observations establish an important role for Egr4 in the porcine ovary.
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Payton, R. R., L. A. Rispoli, and J. L. Edwards. "193 DIFFERENTIAL GENE EXPRESSION IN CUMULUS CELLS OF DEVELOPMENTALLY COMPETENT V. CHALLENGED BOVINE OOCYTES." Reproduction, Fertility and Development 21, no. 1 (2009): 195. http://dx.doi.org/10.1071/rdv21n1ab193.
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It is well established that exposure of cumulus–oocyte complexes (COC) to heat stress during the first 12 h of maturation reduces blastocyst development by 42 to 65%. Previous research supports the notion that some of the effects of heat stress on oocyte competence may be cumulus-mediated. To determine the extent to which this may occur, COC were matured at 38.5°C for 24 h (control) or 41°C for the first 12 h of maturation followed by 38.5°C for remaining 12 h (heat stress). A subset of COC underwent IVF with Percoll-prepared sperm and then was cultured in KSOM containing 0.5% BSA to assess developmental competence. Remaining oocytes were denuded. Cumulus cells, kept separate by treatment, were stored in lysis buffer at –80°C until RNA extraction. Total RNA from cumulus was amplified prior to hybridization to bovine Affymetrix GeneChips (Affymetrix Inc., Santa Clara, CA, USA; n = 8 pools per treatment collected on 8 different occasions; n = 16 chips). Following pre-processing using the MAS5.0 algorithm, microarray data were subjected to linear modeling and empirical Bayes analyses (Bioconductor, Limma package). False discovery rate was controlled using the Benjamini and Hochberg method, and differentially expressed genes were selected by an adjusted P-value (P < 0.05). Functional annotation of selected genes was performed using NetAffx (Affymetrix Inc.) and Database for Annotation, Visualization and Integrated Discovery (DAVID; NIAID, NIH, Bethesda, MD, USA). Heat stress of COC reduced blastocyst development (27.2 v. 16.1% for control v. heat stress, respectively; SEM = 1.6; P < 0.002). Approximately 66 and 65% of 24 000 possible genes were called present (i.e. expressed) in RNA from cumulus of competent (control) v. challenged (heat-stressed) oocytes, respectively. In cumulus from developmentally challenged COC, increased abundance of 42 genes (36 currently annotated) was noted. Use of DAVID demonstrated enrichment of genes important for electron transport and energy generation (NOS2A, MAOB, CYP11A1, HSD11B1L, LTB4DH). Further examination of gene ontology identified genes associated with mitochondrial function (SLC25A10, MAOB, CYP11A1), cell signaling (similar to JAK-3, FSHR, CYP11A1, WNT2B), cytoskeleton (ACTA1), antioxidant activity (GSTA1), and extracellular region (FMOD). In contrast, cumulus from developmentally competent COC had increased expression of 22 genes (20 currently annotated), of which 15% were related to protein binding (CAV1, MMP9, TGFB2) according to DAVID. Further analysis using gene ontology revealed genes associated with extracellular matrix formation (MMP9, MMP19, PCOLCE2) and neural tissue (METRNL). In summary, alterations in cumulus gene expression were associated with differences in developmental competence of oocytes. Additional research is necessary to examine the extent to which identified genes account for functional differences in oocyte competence. This research was supported in part by National Research Initiative Competitive Grant no. 2004-35203-14772 from the USDA Cooperative State Research, Education, and Extension Service.
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Barnett, Melanie J., Alycia N. Bittner, Carol J. Toman, Valerie Oke, and Sharon R. Long. "Dual RpoH Sigma Factors and Transcriptional Plasticity in a Symbiotic Bacterium." Journal of Bacteriology 194, no. 18 (July 6, 2012): 4983–94. http://dx.doi.org/10.1128/jb.00449-12.
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ABSTRACTSinorhizobium melilotican live as a soil saprophyte and can engage in a nitrogen-fixing symbiosis with plant roots. To succeed in such diverse environments, the bacteria must continually adjust gene expression. Transcriptional plasticity in eubacteria is often mediated by alternative sigma (σ) factors interacting with core RNA polymerase. TheS. melilotigenome encodes 14 of these alternative σ factors, including two putative RpoH (“heat shock”) σ factors. We used custom Affymetrix symbiosis chips to characterize the global transcriptional response ofS. melilotirpoH1,rpoH2, andrpoH1 rpoH2mutants during heat shock and stationary-phase growth. Under these conditions, expression of over 300 genes is dependent onrpoH1andrpoH2. We mapped transcript start sites of 69rpoH-dependent genes using 5′ RACE (5′ rapid amplification of cDNA ends), which allowed us to determine putative RpoH1-dependent, RpoH2-dependent, and dual-promoter (RpoH1- and RpoH2-dependent) consensus sequences that were each used to search the genome for other potential direct targets of RpoH. The inferredS. melilotiRpoH promoter consensus sequences share features ofEscherichia coliRpoH promoters but lack extended −10 motifs.
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Condomines, Maud, Dirk Hose, Thierry Reme, John de Vos, Guilhem Requirand, Jean-Francois Rossi, Hartmunt Goldschmidt, and Bernard Klein. "Gene Expression Profiling and Real-Time PCR Analyses Make It Possible To Identify Novel Potential Cancer-Testis Antigens in Multiple Myeloma." Blood 110, no. 11 (November 16, 2007): 1793. http://dx.doi.org/10.1182/blood.v110.11.1793.1793.
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Abstract The identification of novel tumor-associated antigens is critical for the development of immunotherapeutic strategies. Cancer-testis (CT) antigens represent attractive targets due to their restricted pattern of expression. More than 90 CT genes have been previously classified into four categories according to their expression profiles: testis-restricted (expression in testis and tumor samples only), “tissue restricted” (mRNA detected in 2 or fewer non-gametogenic tissues), “differentially expressed” (mRNA detected in three to six non-gametogenic tissues), and “ubiquitously expressed”. Among those, we previously reported that 18 CT genes were expressed by primary myeloma cells (MMC) of more than 10% of patients with multiple myeloma (MM). This study aimed at finding novel putative CT genes expressed in MM using cDNA microarray analysis and real-time RT-PCR validation. Gene expression profiles of 5 testis samples, 64 MMC, 7 normal memory B cell (MB), 7 normal bone marrow plasma cell samples and 23 normal tissue samples available on a public database were obtained using Affymetrix U133AB microarrays. Out of 45000 probe sets of Affymetrix U133 AB chips, we selected 16982 probe sets which had a “Present” Affymetrix Call in MMC of at least 6/64 patients and in 3/5 testis samples. In order to select genes with a similar pattern of expression than the known CT genes, we developed 4 independent filters making it possible to keep a high number of known CT genes while decreasing the total number of probe sets. Firstly, 2514 of 16982 probe sets had a ratio of the mean signal in MMC with a Present call / mean signal in MB > 2.5. Secondly, 541 of these 2514 probe sets had a Present call in less than 7 of the 23 normal tissues. Thirdly, 333 of these 541 probe sets had a ratio of the mean signal in MMC with a Present call / mean signal in MMC with an Absent call > 2.5. Fourthly, we removed genes whose expression profiles were discordant with different probe sets or discordant with data of the literature. The final probe set list contains 88 probe sets which include 13 of 18 known CT genes reported in MM, thus resulting in a 190-fold enrichment. The expression in 13 normal tissues and in MM samples of 21 out of these 75 putative novel CT genes was investigated by real time RT-PCR. Seven genes were ubiquitously expressed or poorly expressed in MMC samples and further deleted. According to the previously defined CT gene categories, we found one novel “testis-restricted” (TEX14), 8 “tissue-restricted” and 5 “differentially expressed” CT genes. Immunogenicity of one gene product - IGSF11 - was already demonstrated in other cancers by identifying a T-cell epitope. Two genes - NLGN4X and FAM133A - are located in X chromosome and 2 genes - CTNNA2 and FAM133A - are expressed only in brain and testis. In conclusion, by analyzing gene expression patterns with Affymetrix microarrays, we found 75 novel putative CT antigen candidates expressed in MMC of 10 to 100% of patients. Real time RT-PCR validation made it possible to confirm the CT status of 14 genes out of the 21 tested. Further studies are warranted to determine their immunogenicity.
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Pellagatti, Andrea, Mario Cazzola, Aristoteles Giagounidis, Luca Malcovati, Matteo G. Della Porta, Martin Jädersten, Sally Killick, et al. "Expression Profiling of CD34+ Cells in Patients with Myelodysplastic Syndromes: Involvement of Interferon-Stimulated Genes and Correlation to FAB Subtype and Karyotype." Blood 108, no. 11 (November 16, 2006): 2626. http://dx.doi.org/10.1182/blood.v108.11.2626.2626.
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Abstract The myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic malignancies. We have used Affymetrix microarray technology to determine the gene expression profiles in CD34+ cells of 84 MDS patients (25 RA, 28 RARS and 31 RAEB) and 16 healthy controls. Twenty-five of 84 patients had a del(5q). CD34+ cells were isolated from bone marrow samples using MACS magnetic columns. Extracted total RNA was amplified using the Two-Cycle Target Labelling kit (Affymetrix) and samples were hybridized to Affymetrix U133 Plus2.0 chips (representing 39,000 human genes). Cell intensity calculation and scaling was performed using GeneChip Operating Software and data analysis using GeneSpring 7.3. The expression profiles of MDS CD34+ cells showed many similarities to reported interferon-γ induced gene expression in normal CD34+ cells. Indeed the two most up-regulated genes, IFIT1 and IFITM1, are interferon-stimulated genes. IFIT1 and IFITM1 were up-regulated by >2-fold in 58/84 and 53/84 MDS patients respectively. Genes down-regulated by >2-fold in the majority of MDS patients include the putative tumor suppressor gene Gravin/AKAP12, ARPP-21, CD24 and MME. The association of distinct gene expression profiles with specific FAB and cytogenetic groups was determined using data from 55 MDS patients as a training set. Hierarchical clustering performed using 457 significantly different genes between different FAB subtypes showed that MDS patients with RARS constitute a homogeneous group, while MDS patients with RA and RAEB show more overlap. CD34+ cells from patients with RARS showed up-regulation of mitochondrial-related genes, and in particular of those of heme synthesis (e.g. ALAS2). Statistical analysis showed that 889 probe-sets could discriminate MDS patients with a del(5q) from those without a del(5q). MDS patients with the del(5q) showed distinctive down-regulation of genes mapping to chromosome 5q, and up-regulation of the histone HIST1 gene cluster at chromosome 6p21 and of genes related to the actin cytoskeleton. In order to identify genes differentially expressed between early and advanced MDS, a comparison was made between the 18 patients with RA and the nine MDS patients with RAEBII. 762 significantly different probe sets were identified that could group together MDS patients with RAEBII. The most significant genes identified include CASP3 and FLT3, and represent potential prognostic markers or markers of disease progression. The remaining 29 MDS patients were used as a test set for class prediction using support vector machines. The FAB subtype was correctly predicted for 83% of the test samples. The presence or absence of a del(5q) was predicted correctly for 93% of the test samples. Finally, 94% of the test samples were predicted correctly as RA or RAEBII. This study provides important and new insights into the pathophysiology of MDS.
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Sukumaran, Siddharth, Bai Xue, William J. Jusko, Debra C. DuBois, and Richard R. Almon. "Circadian variations in gene expression in rat abdominal adipose tissue and relationship to physiology." Physiological Genomics 42A, no. 2 (October 2010): 141–52. http://dx.doi.org/10.1152/physiolgenomics.00106.2010.
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Circadian rhythms occur in all levels of organization from expression of genes to complex physiological processes. Although much is known about the mechanism of the central clock in the suprachiasmatic nucleus, the regulation of clocks present in peripheral tissues as well as the genes regulated by those clocks is still unclear. In this study, the circadian regulation of gene expression was examined in rat adipose tissue. A rich time series involving 54 animals euthanized at 18 time points within the 24-h cycle (12:12 h light-dark) was performed. mRNA expression was examined with Affymetrix gene array chips and quantitative real-time PCR, along with selected physiological measurements. Transcription factors involved in the regulation of central rhythms were examined, and 13 showed circadian oscillations. Mining of microarray data identified 190 probe sets that showed robust circadian oscillations. Circadian regulated probe sets were further parsed into seven distinct temporal clusters, with >70% of the genes showing maximum expression during the active/dark period. These genes were grouped into eight functional categories, which were examined within the context of their temporal expression. Circadian oscillations were also observed in plasma leptin, corticosterone, insulin, glucose, triglycerides, free fatty acids, and LDL cholesterol. Circadian oscillation in these physiological measurements along with the functional categorization of these genes suggests an important role for circadian rhythms in controlling various functions in white adipose tissue including adipogenesis, energy metabolism, and immune regulation.
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Dmitriev, Alexander V., Emily J. McDowell, Kyle V. Kappeler, Michelle A. Chaussee, Lindsey D. Rieck, and Michael S. Chaussee. "The Rgg Regulator of Streptococcus pyogenes Influences Utilization of Nonglucose Carbohydrates, Prophage Induction, and Expression of the NAD-Glycohydrolase Virulence Operon." Journal of Bacteriology 188, no. 20 (October 1, 2006): 7230–41. http://dx.doi.org/10.1128/jb.00877-06.
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ABSTRACT The expression of many virulence-associated genes in Streptococcus pyogenes is controlled in a growth phase-dependent manner. Unlike the model organisms Escherichia coli and Bacillus subtilis, such regulation is apparently not dependent upon alternative sigma factors but appears to rely on complex interactions among several transcriptional regulators, including Rgg. The purpose of this study was to identify changes in gene expression associated with inactivation of the rgg gene in S. pyogenes strain NZ131 (serotype M49). To this end, the transcriptomes of wild-type and rgg mutant strains were analyzed during both the exponential and postexponential phases of growth using Affymetrix NimbleExpress gene chips. Genomewide differences in transcript levels were identified in both phases of growth. Inactivation of rgg disrupted coordinate expression of genes associated with the metabolism of nonglucose carbon sources, such as fructose, mannose, and sucrose. The changes were associated with an inability of the mutant strain to grow using these compounds as the primary carbon source. Bacteriophage transcript levels were also altered in the mutant strain and were associated with decreased induction of at least one prophage. Finally, transcripts encoding virulence factors involved in cytolysin-mediated translocation of NAD-glycohydrolase, including the immunity factor IFS and the cytolysin (streptolysin O [SLO]), were more abundant in the mutant strain, which correlated with the amount of NADase and SLO activities in culture supernatant fluids. The results provide further evidence that Rgg contributes to growth phase-dependent gene regulation in strain NZ131.
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Sun, Yan, Dong Li, Sumanprava Giri, Supriya G. Prasanth, and Dongwan Yoo. "Differential Host Cell Gene Expression and Regulation of Cell Cycle Progression by Nonstructural Protein 11 of Porcine Reproductive and Respiratory Syndrome Virus." BioMed Research International 2014 (2014): 1–13. http://dx.doi.org/10.1155/2014/430508.
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Nonstructural protein 11 (nsp11) of porcine reproductive and respiratory syndrome virus (PRRSV) is a viral endoribonuclease with an unknown function. The regulation of cellular gene expression by nsp11 was examined by RNA microarrays using MARC-nsp11 cells constitutively expressing nsp11. In these cells, the interferon-β, interferon regulatory factor 3, and nuclear factor-κB activities were suppressed compared to those of parental cells, suggesting that nsp11 might serve as a viral interferon antagonist. Differential cellular transcriptome was examined using Affymetrix exon chips representing 28,536 transcripts, and after statistical analyses 66 cellular genes were shown to be upregulated and 104 genes were downregulated by nsp11. These genes were grouped into 5 major signaling pathways according to their functional relations: histone-related, cell cycle and DNA replication, mitogen activated protein kinase signaling, complement, and ubiquitin-proteasome pathways. Of these, the modulation of cell cycle by nsp11 was further investigated since many of the regulated genes fell in this particular pathway. Flow cytometry showed that nsp11 caused the delay of cell cycle progression at the S phase and the BrdU staining confirmed the cell cycle arrest in nsp11-expressing cells. The study provides insights into the understanding of specific cellular responses to nsp11 during PRRSV infection.
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Knight, Pamela A., Alan D. Pemberton, Kevin A. Robertson, Douglas J. Roy, Steven H. Wright, and Hugh R. P. Miller. "Expression Profiling Reveals Novel Innate and Inflammatory Responses in the Jejunal Epithelial Compartment during Infection with Trichinella spiralis." Infection and Immunity 72, no. 10 (October 2004): 6076–86. http://dx.doi.org/10.1128/iai.72.10.6076-6086.2004.
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ABSTRACT Infection with intestinal nematodes induces profound pathological changes to the gut that are associated with eventual parasite expulsion. We have applied expression profiling as an initial screening process with oligonucleotide microarrays (Affymetrix MG-U74AV2 gene chips) and time course kinetics to investigate gene transcription triggered by the intraepithelial nematode Trichinella spiralis in jejunal epithelium from BALB/c mice. Of the 4,114 genes detected, 2,617 were present in all uninfected and T. spiralis-infected replicates, 8% of which were notably upregulated, whereas 12% were downregulated at the time of worm expulsion (day 14 postinfection). Upregulation of goblet cell mucin gene transcripts intestinal mucin gene 3 (MUC3), calcium chloride channel 5 (CLCA5), and goblet cell gene 4 (GOB4) is consistent with enhanced production and alteration of mucus, whereas a 60- to 70-fold upregulation of transcripts for mast cell proteases 1 and 2 (MCPT-1 and -2) is consistent with intraepithelial mucosal mast cell recruitment. Importantly, there was novel expression of sialyltransferase 4C (SIAT4C), small proline-rich protein 2A (SPRR2A), and resistin-like molecule β (RELMβ) on day 14 postinfection. In contrast, DNase I and regenerating protein 3 (REG3) transcripts were substantially downregulated. Time course analyses revealed early (within 48 h of infection) induction of Siat4c, Sprr2A, and Relmβ and later (within 120 h) induction of Mcpt-1 and -2. The findings demonstrate early innate responses and later inflammatory changes within the epithelium. The early epithelial responses may be associated both with repair (Sprr2A) and with the development of innate immunity (Siat4c and Relmβ).
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Poulain, Stephanie, Rachid Aijjou, Natacha Broucqsault, Salomon Manier, Agnes Daudignon, Charles Herbaux, Sylvie Galiegue-Zouitina, et al. "A20 Gene Deregulation In Waldenstrom's Macroglobulinemia." Blood 116, no. 21 (November 19, 2010): 3628. http://dx.doi.org/10.1182/blood.v116.21.3628.3628.
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Abstract Abstract 3628 Background. Waldenstrom's macroglobulinemia (WM) is a rare lymphoproliferative disorder characterized by bone marrow (BM) infiltration of lymphoplasmacytic cells and monoclonal IgM gammopathy. The deletion 6q is the most frequent cytogenetic aberration in WM and it has been suspected that this region harbours a tumour suppressor gene of pathogenic significance for WM. Comparative genomic hybridization (CGH) array delineated several minimal deleted regions (MDR) on 6q and pointed out key regulator genes of the NFKB pathway involved in these deletions, including A20 gene. The zinc finger protein A20 has been characterized as dual inhibitor of NFKB activation and was recently described as a tumor suppressor gene in lymphoma. However, the mechanisms of A20 deregulation are not fully understood in WM. We aimed to study the mechanisms of A20 deregulation in WM using gene expression profiling (GEP) and single nucleotide polymorphism (SNP) based arrays, a powerful high resolution method allowing both the detection of loss of heterozygosity (LOH) and copy number alteration (CNA) analysis in the same experiment. Methods. We have studied BM samples from 42 patients (pts) with WM (31 males, mean age: 67 years). Conventional karyotype study was analyzed following stimulation with IL2 and DSP30 of BM cells. FISH analysis was performed to detect deletion 6q21 (MYB probe). DNA and RNA were extracted following BM CD19 B cell negative selection. Quantification of copy number of A20 gene was performed using real time PCR, RNAse P gene was used as reference gene. Genome-Wide Human SNP Array 6.0 (Affymetrix chips) was used to detect both LOH and CNA in 31 pts. Paired samples (B cells/normal T lymphocytes) were used as an intra-individual reference to distinguish germline polymorphisms from somatic abnormalities in 29 patients. Size, position and location of genes were referenced using UCSC HG18 assembly, LOH and CNA were identified using genotyping console 3.02 software (Affymetrix) and Partek genomic suite. Gene expression profiling (GEP) was performed in 11 pts using Affymetrix U133A arrays. Gene-expression intensity values were log transformed, normalized and analyzed using GeneSpring GX software. Results. SNP array identified deletion 6q in 9/31 (29%) patients, confirmed by FISH analysis. All cases were monoallelic deletion. In 90% of cases, deletion 6q was associated with others genetic abnormalities. In one case, we observed a loss of 6q combined with a gain of 6p. Deletion 6q was associated with gain of chromosome 4 in 2 cases, and with deletion of 13q in 3 cases. The MDR was located on chromosome 6q21-6q25. This MDR contained several candidate genes included always deletion of A20 gene, located on 6q23, and was confirmed by real time DNA PCR quantification in all cases. Overall, the frequency of A20 gene deletion in our entire WM cohort (N=42 pts) was 29.2% (12/42), determined by real time DNA PCR of the copy number of A20 gene. In patient without deletion 6q, we looked at potential deregulated mechanisms of A20 by LOH with no CNA, e.g. UPD (uniparental disomy). We have not seen any UPD targeting the A20 gene. We then looked at A20 gene expression by GEP, and found no significant variation of A20 gene expression related to A20 monoallelic deletion as compared to pts with two copies, reflecting probably the existence of other mechanisms of A20 gene deregulation. This result was consistent with the absence of clear difference in genome-wide expression profiling in pts with 6q deletion and those without the deletion. As A20 is a key player in the negative feedback loop regulation of NFKB, we also looked at the gene expression of a set of genes involved in NFKB pathway. The presence or absence of A20 deletion did not influence the gene expression profiling of this NFKB pathway gene set. Conclusion. We described a high frequency of deletion of A20 gene. In most cases, deletion of A20 was associated with other genetic abnormalities. A20 deletion was not associated with a significant signature by GEP. Additional studies are needed to understand the cellular consequences of A20 deletions in the pathogenesis of WM. Disclosures: Leleu: Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.
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Sonna, Larry A., Matthew M. Kuhlmeier, Heather C. Carter, Jeffrey D. Hasday, Craig M. Lilly, and Karen D. Fairchild. "Effect of moderate hypothermia on gene expression by THP-1 cells: a DNA microarray study." Physiological Genomics 26, no. 1 (June 2006): 91–98. http://dx.doi.org/10.1152/physiolgenomics.00296.2005.
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The mechanisms by which moderate hypothermia (32°C for 12–72 h) affect human cellular function are unclear. We tested the hypothesis that it produces broad changes in mRNA expression in vitro. Acute monocytic leukemia (THP-1) cells were incubated under control conditions (37°C) or moderate hypothermia (32°C) for 24 h. RNA was extracted, and the hypothermic response was confirmed by examining the expression of the cold-induced RNA-binding protein (CIRBP) gene by RT-PCR. Gene expression analysis was performed on seven sets of paired samples with Affymetrix U133A chips using established statistical methods. Sequences were considered affected by cold if they showed statistically significant changes in expression and also met published post hoc filter criteria (changes in geometric mean expression of ≥2-fold and expression calls of “present” or “marginal” in at least half of the experiments). Changes in the expression of selected sequences were further confirmed by PCR. Sixty-seven sequences met the criteria for increased expression (including cold-inducible genes CIRBP and RNA binding motif 3), and 100 sequences showed decreased expression as a result of hypothermia. Functional categories affected by hypothermia included genes involved in immune responses; cell growth, proliferation, and differentiation; and metabolism and biosynthesis. Several heat shock proteins (HSPs) showed decreases in expression. Moderate hypothermia produces substantial changes in gene expression, in categories potentially of systemic importance. Cold exposure without rewarming decreased the expression of several HSPs. These in vitro findings suggest that prolonged hypothermia in vivo might be capable of producing physiologically relevant changes in gene expression by circulating leukocytes.
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Cho, Hyung Jun, Pavel Shashkin, Christian A. Gleissner, Dane Dunson, Nitin Jain, Jae K. Lee, Yury Miller, and Klaus Ley. "Induction of dendritic cell-like phenotype in macrophages during foam cell formation." Physiological Genomics 29, no. 2 (April 2007): 149–60. http://dx.doi.org/10.1152/physiolgenomics.00051.2006.
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Foam cell formation from monocyte-derived macrophages is a hallmark of atherosclerotic lesions. Aspects of this process can be recapitulated in vitro by exposing M-CSF-induced or platelet factor 4 (CXCL4)-induced macrophages to oxidized (ox) or minimally modified (mm) low density lipoprotein (LDL). We measured gene expression in peripheral blood mononuclear cells, monocytes, and macrophages treated with CXCL1 (GRO-α) or CCL2 (MCP-1), as well as foam cells induced by native LDL, mmLDL, or oxLDL using 22 Affymetrix gene chips. Using an advanced Bayesian error-pooling approach and a heterogeneous error model with a false discovery rate <0.05, we found 5,303 of 22,215 probe sets to be significantly regulated in at least one of the conditions. Among a subset of 917 candidate genes that were preselected for their known biological functions in macrophage foam-cell differentiation, we found that 290 genes met the above statistical criteria for significant differential expression patterns. While many expected genes were found to be upregulated by LDL and oxLDL, very few were induced by mmLDL. We also found induction of unexpected genes, most strikingly MHC-II and other dendritic cell markers such as CD11c. The gene expression patterns in response to oxLDL were similar in M-CSF-induced and CXCL4-induced macrophages. Our findings suggest that LDL and oxLDL, but not mmLDL, induce a dendritic cell-like phenotype in macrophages, suggesting that these cells may be able to present antigens and support an immune response.
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Esteva, F. J., K. Anderson, F. Lin, R. Nahta, J. Mejia, K. Altundag, A. U. Buzdar, G. N. Hortobagyi, W. F. Symmans, and L. Pusztai. "Pharmacogenomic analysis of HER2 amplified breast cancer treated with preoperative trastuzumab and paclitaxel, 5-fluorouracil, epirubicin, cyclophosphamide (T/FEC) chemotherapy." Journal of Clinical Oncology 24, no. 18_suppl (June 20, 2006): 545. http://dx.doi.org/10.1200/jco.2006.24.18_suppl.545.
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545 Background: We performed gene expression analysis to identify molecular predictors of resistance to preoperative concomitant trastuzumab and T/FEC chemotherapy. Methods: Pretreatment fine needle aspirations from 21 patients with HER2 amplified, stages II-III breast cancer were available for transcriptional profiling with Affymetrix U133 A chips. Results: Overall pathologic complete response (pCR) rate was 71%. Age, nuclear grade, tumor size, nodal status or quantitative estrogen and HER2 receptor mRNA expression showed no association with response in univariate and multivariate logistic regression. We tested the accuracy of a 30-gene pCR predictor that was previously developed from patients who received T/FEC only preoperative chemotherapy. This predictor was accurate in validation in T/FEC treated patients (n=51) but showed low sensitivity in patients who also received trastuzumab (sensitivity 53% versus 92%). We could not identify any differentially expressed genes between pCR (n=15) and residual disease (RD, n=6) at a false discovery rate (FDR) <90% in the HER2 amplified trastuzumab-treated cases. Hierarchical clustering using the “Perou intrinsic gene set” also failed to separate pCR from RD. Gene Set Enrichment Analysis with 22 genes from trastuzumab-resistant cell lines showed a modest association with RD (FDR=9%). Conclusions: Clinical variables and pharmacogenomic predictors that predict pCR in the absence of trastuzumab are no longer accurate when trastuzumab is included in the treatment. Trastuzumab-resistance associated genes identified in vitro are also associated with resistance in vivo. Support: Ellence Foundation. No significant financial relationships to disclose.
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Reppe, Sjur, Lis Stilgren, Bo Abrahamsen, Ole K. Olstad, Fadila Cero, Kim Brixen, Lise Sofie Nissen-Meyer, and Kaare M. Gautvik. "Abnormal muscle and hematopoietic gene expression may be important for clinical morbidity in primary hyperparathyroidism." American Journal of Physiology-Endocrinology and Metabolism 292, no. 5 (May 2007): E1465—E1473. http://dx.doi.org/10.1152/ajpendo.00487.2006.
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In primary hyperparathyroidism (PHPT), excess PTH secretion by adenomatous or hyperplastic parathyroid glands leads to elevated serum [Ca2+]. Patients present complex symptoms of muscular fatigue, various neuropsychiatric, neuromuscular, and cardiovascular manifestations, and, in advanced disease, kidney stones and metabolic bone disease. Our objective was to characterize changes in muscle and hematopoietic gene expression in patients with reversible mild PHPT after parathyroidectomy and possibly link molecular pathology to symptoms. Global mRNA profiling using Affymetrix gene chips was carried out in biopsies obtained before and 1 yr after parathyroidectomy in seven patients discovered by routine blood [Ca2+] screening. The tissue distribution of PTH receptor (PTHR1 and PTHR2) mRNAs were quantitated using real-time RT-PCR in unrelated persons to define PTH target tissues. Of about 10,000 expressed genes, 175 muscle, 169 hematological, and 99 bone-associated mRNAs were affected. Notably, the major part of muscle-related mRNAs was increased whereas hematological mRNAs were predominantly decreased during disease. Functional and molecular network analysis demonstrated major alterations of several tissue characteristic groups of mRNAs as well as those belonging to common cell signaling and major metabolic pathways. PTHR1 and PTHR2 mRNAs were more abundantly expressed in muscle and brain than in hematopoietic cells. We suggest that sustained stimulation of PTH receptors present in brain, muscle, and hematopoietic cells have to be considered as one independent, important cause of molecular disease in PHPT leading to profound alterations in gene expression that may help explain symptoms like muscle fatigue, cardiovascular pathology, and precipitation of psychiatric illness.
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Fonseca, Rafael, Scott Van Wier, Wee-Joo Chng, Rhett Ketterling, Martha Lacy, Angela Dispenzieri, Peter Leif Bergsagel, et al. "Low Level Amplification (Duplication) of 1q21 in Myeloma and Prognosis; the Role of CKS1B." Blood 106, no. 11 (November 16, 2005): 624. http://dx.doi.org/10.1182/blood.v106.11.624.624.
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Abstract Introduction: Molecular cytogenetic studies have revealed that, to a great extent, the heterogeneity of myeloma is largely dictated by the underlying genetic and cytogenetic aberrations present in the clonal plasma cells. Most recently the group from the University of Arkansas identified strong prognostic associations with an increased level of gene expression of a cell cycle associated gene, CKS1B. CKS1B favors cell cycle progression by promoting degradation of p27 with release of the cyclin dependent kinases and entry into mitosis. Patients and methods: To further test this hypothesis we studied: a) via FISH for CKS1B amplification in a cohort of patients treated at the Mayo Clinic with high dose chemotherapy and stem cell support, as well as a group of patients with cytogenetically defined hypodiploidy, and b) a cohort of myeloma patients that were studied by gene expression profiling. Gene expression analysis was performed on CD138-enriched plasma-cell RNA using Affymetrix U133A chips (Affymetrix, Santa Clara, CA). Results: Of 159 patients studied 46 exhibited FISH abnormalities consistent with increase number of signals for CKS1B (30%): amplification was marginal or low in 44 cases, and in only two cases the ratio between CKS1B and control probe was greater than 2.0. Therefore, the predominant pattern is one of gene duplication rather than amplification where gene copy usually exceeds 10 and ratio with control probe exceeds 5 (e.g. HER-2/neu amplification). Patients with CKS1B duplication had a higher prevalence of chromosome 13 deletion (72%), and t(4;14) (29%). The presence of CKS1B duplication was more frequent (53%) among 19 patients with hypodiploidy. Using gene expression data for CKS1B, we found a positive association with t(14;16)(q32;q23). The level of CKS1B gene expression positively correlated with the PCLI (p<0.0001) but there was heterogeneity in this relationship (r2 = 0.34). We found no correlation between the expression level of CKS1B and p27 (r2 = 0.008, p = 0.37). While CKS1B gene duplication had a negative effect on survival this effect was weak (29.9 versus 38 months, p = 0.124. Median follow-up is 55 months) and disappeared when the variable was entered into the multivariate model including PCLI, B2-microglobulin and all the major genetic abnormalities by FISH. Likewise the level of expression of CKS1B determined by gene expression only carried prognostic significance when extreme levels of expression were utilized as a prognostic variable. Discussion: In this study we show that increase copy number of CKS1B is present in one third of patients with MM (majority of these being gene duplication) and seem to be associated with a shorter overall survival, but the net effect of this is rather weak in our series. Further study is needed to understand its potential role as a progression factor in the plasma cell neoplasms.
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Sonna, Larry A., C. Bruce Wenger, Scott Flinn, Holly K. Sheldon, Michael N. Sawka, and Craig M. Lilly. "Exertional heat injury and gene expression changes: a DNA microarray analysis study." Journal of Applied Physiology 96, no. 5 (May 2004): 1943–53. http://dx.doi.org/10.1152/japplphysiol.00886.2003.
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This study examined gene expression changes associated with exertional heat injury (EHI) in vivo and compared these changes to in vitro heat shock responses previously reported by our laboratory. Peripheral blood mononuclear cell (PBMC) RNA was obtained from four male Marine recruits (ages 17-19 yr) who presented with symptoms consistent with EHI, core temperatures ranging from 39.3 to 42.5°C, and elevations in serum enzymes such as creatine kinase. Controls were age- and gender-matched Marines from whom samples were obtained before and several days after an intense field-training exercise in the heat (“The Crucible”). Expression analysis was performed on Affymetrix arrays (containing ∼12,600 sequences) from pooled samples obtained at three times for EHI group (at presentation, 2-3 h after cooling, and 1-2 days later) and compared with control values (average signals from two chips representing pre- and post-Crucible samples). After post hoc filtering, the analysis identified 361 transcripts that had twofold or greater increases in expression at one or more of the time points assayed and 331 transcripts that had twofold or greater decreases in expression. The affected transcripts included sequences previously shown to be heat-shock responsive in PBMCs in vitro (including both heat shock proteins and non-heat shock proteins), a number of sequences whose changes in expression had not previously been noted as a result of in vitro heat shock in PBMCs (including several interferon-induced sequences), and several nonspecific stress response genes (including ubiquitin C and dual-specificity phosphatase-1). We conclude that EHI produces a broad stress response that is detectable in PBMCs and that heat stress per se can only account for some of the observed changes in transcript expression. The molecular evidence from these patients is thus consistent with the hypothesis that EHI can result from cumulative effects of multiple adverse interacting stimuli.
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Collins, James F., Zihua Hu, P. N. Ranganathan, Dian Feng, Laura M. Garrick, Michael D. Garrick, and Richard W. Browne. "Induction of arachidonate 12-lipoxygenase (Alox15) in intestine of iron-deficient rats correlates with the production of biologically active lipid mediators." American Journal of Physiology-Gastrointestinal and Liver Physiology 294, no. 4 (April 2008): G948—G962. http://dx.doi.org/10.1152/ajpgi.00274.2007.
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To identify novel genes associated with iron metabolism, we performed gene chip studies in two models of iron deficiency: iron-deprived rats and rats deficient in the principal intestinal iron transporter, divalent metal transporter 1 (i.e., Belgrade rats). Affymetrix rat genome gene chips were utilized (RAE230) with cRNA samples derived from duodenum and jejunum of experimental and control animals. Computational analysis and statistical data reduction identified 29 candidate genes, which were induced in both models of iron deficiency. Gene ontology analysis showed enrichment for genes related to lipid homeostasis, and one gene related to this physiological process, a leukocyte type, arachidonate 12-lipoxygenase ( Alox15), was selected for further examination. TaqMan real-time PCR studies demonstrated strong induction of Alox15 throughout the small and large intestine, and in the liver of iron-deficient rats. Polyclonal antibodies were developed and utilized to demonstrate that proteins levels are significantly increased in the intestinal epithelium of iron-deprived rats. HPLC analysis revealed altered intestinal lipid metabolism indicative of Alox15 activity, which resulted in the production of biologically active lipid molecules (12-HETE, 13-HODE, and 13-HOTE). The overall effect is a perturbation of intestinal lipid homeostasis, which results in the production of lipids essentially absent in the intestine of control rats. We have thus provided mechanistic insight into the alteration in lipid metabolism that occurs during iron deficiency, in that induction of Alox15 mRNA expression may be the primary event. The resulting lipid mediators may be related to documented alterations in villus structure and cell proliferation rates in iron deficiency, or to structural alterations in membrane lipid composition.
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McAleese, Fionnuala, Shang Wei Wu, Krzysztof Sieradzki, Paul Dunman, Ellen Murphy, Steven Projan, and Alexander Tomasz. "Overexpression of Genes of the Cell Wall Stimulon in Clinical Isolates of Staphylococcus aureus Exhibiting Vancomycin-Intermediate- S. aureus-Type Resistance to Vancomycin." Journal of Bacteriology 188, no. 3 (February 1, 2006): 1120–33. http://dx.doi.org/10.1128/jb.188.3.1120-1133.2006.
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ABSTRACT Custom-designed gene chips (Affymetrix) were used to determine genetic relatedness and gene expression profiles in Staphylococcus aureus isolates with increasing MICs of vancomycin that were recovered over a period of several weeks from the blood and heart valve of a patient undergoing extensive vancomycin therapy. The isolates were found to be isogenic as determined by the GeneChip based genotyping approach and thus represented a unique opportunity to study changes in gene expression that may contribute to the vancomycin resistance phenotype. No differences in gene expression were detected between the parent strain, JH1, and JH15, isolated from the nares of a patient contact. Few expression changes were observed between blood and heart valve isolates with identical vancomycin MICs. A large number of genes had altered expression in the late stage JH9 isolate (MIC = 8 μg/ml) compared to JH1 (MIC = 1 μg/ml). Most genes with altered expression were involved in housekeeping functions or cell wall biosynthesis and regulation. The sortase-encoding genes, srtA and srtB, as well as several surface protein-encoding genes were downregulated in JH9. Two hypothetical protein-encoding genes, SAS016 and SA2343, were dramatically overexpressed in JH9. Interestingly, 27 of the genes with altered expression in JH9 grown in drug-free medium were found to be also overexpressed when the parental strain JH1 was briefly exposed to inhibitory concentrations of vancomycin, and more than half (17 of 27) of the genes with altered expression belonged to determinants that were proposed to form part of a general cell wall stress stimulon (S. Utaida et al., Microbiology 149:2719-2732, 2003).
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Oswald, Joachim, Christine Steudel, Katrin Salchert, Christian Thiede, Gerhard Ehninger, Carsten Werner, Ulrich Schuler, and Martin Bornhaeuser. "Gene-Chip Analysis of Cord Blood Derived CD34+ Cells Expanded on Bioartificial Materials." Blood 104, no. 11 (November 16, 2004): 4131. http://dx.doi.org/10.1182/blood.v104.11.4131.4131.
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Abstract Expansion of hematopoietic stem cells from neonatal cord blood is an important issue for clinical uses since the number of CD34+ cells in individual cord blood samples is limited and often not sufficient for a successful engraftment in adult individuals. In vivo, hematopoietic stem cells reside in the bone marrow in close vicinity to stromal cells and extracellular matrix molecules. We have established a culture system for the ex vivo expansion of CD34+ cord blood cells utilizing fibrillar collagen 1 as a bioartificial matrix to enable cellular adhesion during cell culture. CD34+ hematopoietic stem cells were isolated via immunomagnetic separation from umbilical cord blood after informed consent and cultivated in presence of recombinant cytokines and reconstituted collagen 1 fibrils as matrix. After seven days of cultivation, expansion of cells, expression of surface molecules cells and expansion of colony forming units were assessed. Additionally gene expression profiling was performed with Affymetrix HG U133A chips interrogating 22,253 probe sets. As control, CD34+ cells were expanded in liquid culture without fibrillar collagen. The overall expansion of CD34+ cells was 4.2 fold + 1.7 compared to 11.1 fold + 2.9 for the control sample. The number of colony forming units (CFU) was increased in the collagen 1 containing samples was elevated (65.1 + 10.3 compared to 26.1 + 7.6 in the control). Gene expression analysis with chip technology showed up regulation of several cytokines (e.g. interleukin 8, interleukin 1a) and also of transcription factors with antiproliferative features like BTG2. The chip data have been verified with quantitative PCR using the Taqman technology. Our data support the idea that direct contact of CD34+ cells with fibrillar collagen 1 results in a delay in cell cycle progression which prevents a subsequent differentiation into more committed progenitors. Therefore fibrillar collagen 1 may serve as supportive matrix for the ex vivo expansion of cord blood derived CD34+ cells.
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Sukumaran, Siddharth, William J. Jusko, Debra C. DuBois, and Richard R. Almon. "Light-dark oscillations in the lung transcriptome: implications for lung homeostasis, repair, metabolism, disease, and drug action." Journal of Applied Physiology 110, no. 6 (June 2011): 1732–47. http://dx.doi.org/10.1152/japplphysiol.00079.2011.
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Diurnal-nocturnal, or circadian-like, rhythms are 24-h variations in biological processes, evolved for the efficient functioning of living organisms. Such oscillations and their regulation in many peripheral tissues are still unclear. In this study, we used Affymetrix gene chips in a rich time-series experiment involving 54 animals killed at 18 time points within the 24-h cycle to examine light-dark cycle patterns of gene expression in rat lungs. Data mining identified 646 genes (represented by 1,006 probe sets) showing robust oscillations in expression in lung that were parsed into 8 distinct temporal clusters. Surprisingly, more than two-thirds of the probe sets showing cyclic expression peaked during the animal's light/inactive period. Six core clock genes and nine clock-related genes showed rhythmic oscillations in their expression in lung. Many of the genes that peaked during the inactive period included genes related to extracellular matrix, cytoskeleton, and protein processing and trafficking, which appear to be mainly involved in the repair and remodeling of the organ. Genes coding for growth factor ligands and their receptors, which play important roles in maintaining normal lung function, also showed rhythmic expression. In addition, genes involved in the metabolism and transport of endogenous compounds, xenobiotics, and therapeutic drugs, along with genes that are biomarkers or potential therapeutic targets for many lung diseases, also exhibited 24-h cyclic oscillations, suggesting an important role for such rhythms in regulating various aspects of the physiology and pathophysiology of lung.
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Huang, Yong, Jian Yan, Ronald Lubet, Thomas W. Kensler, and Thomas R. Sutter. "Identification of novel transcriptional networks in response to treatment with the anticarcinogen 3H-1,2-dithiole-3-thione." Physiological Genomics 24, no. 2 (February 2006): 144–53. http://dx.doi.org/10.1152/physiolgenomics.00258.2005.
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3 H-1,2-dithiole-3-thione (D3T), an inducer of antioxidant and phase 2 genes, is known to enhance the detoxification of environmental carcinogens, prevent neoplasia, and elicit other protective effects. However, a comprehensive view of the regulatory pathways induced by this compound has not yet been elaborated. Fischer F344 rats were gavaged daily for 5 days with vehicle or D3T (0.3 mmol/kg). The global changes of gene expression in liver were measured with Affymetrix RG-U34A chips. With the use of functional class scoring, a semi-supervised method exploring both the expression pattern and the functional annotation of the genes, the Gene Ontology classes were ranked according to the significance of the impact of D3T treatment. Two unexpected functional classes were identified for the D3T treatment, cytosolic ribosome constituents with 90% of those genes increased, and cholesterol biosynthesis with 91% of the genes repressed. In another novel approach, the differentially expressed genes were evaluated by the Ingenuity computational pathway analysis tool to identify specific regulatory networks and canonical pathways responsive to D3T treatment. In addition to the known glutathione metabolism pathway ( P = 0.0011), several other significant pathways were also revealed, including antigen presentation ( P = 0.000476), androgen/estrogen biosynthesis ( P = 0.000551), fatty acid ( P = 0.000216), and tryptophan metabolism ( P = 0.000331) pathways. These findings showed a profound impact of D3T on lipid metabolism and anti-inflammatory/immune-suppressive response, indicating a broader cytoprotective effect of this compound than previously expected.
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Winter, Stuart S., Hadya Khawaja, Zeyu Jiang, Timothy Griffin, Barbara Asselin, and Richard S. Larson. "Identification of Genomic Classifiers That Distinguish Induction Failure in T-Lineage Acute Lymphoblastic Leukemia in COG Study 9404." Blood 108, no. 11 (November 16, 2006): 1826. http://dx.doi.org/10.1182/blood.v108.11.1826.1826.
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Abstract The clinical features of age, white count, and presence of extramedullary disease cannot predict risk for induction failure (IF) in patients who present with T-cell acute lymphoblastic leukemia (T-ALL). On the basis of recent observations that gene expression profiles can distinguish clinicopathologic cohorts of patients with acute leukemia, we hypothesized that microarray analyses performed on diagnostic T-ALL bone marrow samples might identify a genomic classifier for IF patients. Using a case-control study design for children and young adults treated for T-ALL on Children’s Oncology Group Study 9404, we analyzed 50 cryopreserved T-ALL samples using Affymetrix U133A Plus 2 genechips, which have 54,000 genes, ESTs and genomic classifiers. Following RMA normalization, we used Prognostic Multi-array Analysis (PAM) to identify a 116-member genomic classifier that could accurately identify all 6 IF cases from the 44 patients who achieved remission. Within the IF cohort, 37 genes were up-regulated and 79 were down-regulated in comparison to other outcome groups. To further investigate the genetic mechanisms governing IF, we developed four cell lines with acquired drug resistance: Jurkat and Sup T1; each having resistance to daunorubicin (DNR) and asparaginase (ASP). Using a comparative analysis for fold-change in gene expression among 6 IF patients and the T-ALL DNR and ASP-resistant cell lines, we identified seven genes that were up-regulated, and another set of seven genes that were commonly down-regulated. To validate the potential use of our 116-member gene set in predicting IF in T-ALL, we tested our genomic classifier in 42 cases which were treated on COG study 8704 and hybridized to the Affymetrix U133Av.2 chip. Because only 85 probes were shared between U133A Plus 2 and U133Av. 2 chips, we employed shrunken class centroids to constrain our classifier to 25 rank-ordered probes. This smaller classifier correctly identified the single IF case in 8704, as well as another patient who was an early treatment failure, indicating that similar genomic classifiers may identify IF patients in different clinical trials. These results indicate that genetic profiling may be useful in prospectively identifying IF patients in T-ALL. In addition, we identified genes that were commonly upregulated in IF patients and T-ALL cell lines with intrinsic drug resistance.
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Collins, James F., Christina A. Franck, Kris V. Kowdley, and Fayez K. Ghishan. "Identification of differentially expressed genes in response to dietary iron deprivation in rat duodenum." American Journal of Physiology-Gastrointestinal and Liver Physiology 288, no. 5 (May 2005): G964—G971. http://dx.doi.org/10.1152/ajpgi.00489.2004.
Full textAbstract:
We sought to identify novel genes involved in intestinal iron absorption by inducing iron deficiency in rats during postnatal development from the suckling period through adulthood. We then performed comparative gene chip analyses (RAE230A and RAE230B chips; Affymetrix) with cRNA derived from duodenal mucosa. Real-time PCR was used to confirm changes in gene expression. Genes encoding the apical iron transport-related proteins [ divalent metal transporter 1 (DMT1) and duodenal cytochrome b] were strongly induced at all ages studied, whereas increases in mRNA encoding the basolateral proteins iron-regulated gene 1 and hephaestin were observed only by real-time PCR. In addition, transferrin receptor 1 and heme oxygenase 1 were induced. We also identified induction of novel genes not previously associated with intestinal iron transport. The Menkes copper ATPase (ATP7a) and metallothionein were strongly induced at all ages studied, suggesting increased copper absorption by enterocytes during iron deficiency. We also found significantly increased liver copper levels in 7- to 12-wk-old iron-deficient rats. Also upregulated at most ages examined were the sodium-dependent vitamin C transporter, tripartite motif protein 27, aquaporin 4, lipocalin-interacting membrane receptor, and the breast cancer-resistance protein (ABCG2). Some genes also showed decreased expression with iron deprivation, including several membrane transporters, metabolic enzymes, and genes involved in the oxidative stress response. We speculate that dietary iron deprivation leads to increased intestinal copper absorption via DMT1 on the brush-border membrane and the Menkes copper ATPase on the basolateral membrane. These findings may thus explain copper loading in the iron-deficient state. We also demonstrate that many other novel genes may be differentially regulated in the setting of iron deprivation.