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1.  Pharmacogenomic responses of rat liver to methylprednisolone: An approach to mining a rich microarray time series 
The AAPS Journal  2005;7(1):E156-E194.
A data set was generated to examine global changes in gene expression in rat liver over time in response to a single bolus dose of methylprednisolone. Four control animals and 43 drug-treated animals were humanely killed at 16 different time points following drug administration. Total RNA preparation from the livers of these animals were hybridized to 47 individual Affymetrix RU34A gene chips, generating data for 8799 different probe sets for each chip. Data mining techniques that are applicable to gene array time series data sets in order to identify drug-regulated changes in gene expression were applied to this data set. A series of 4 sequentially applied filters were developed that were designed to eliminate probe sets that were not expressed in the tissue, were not regulated by the drug treatment, or did not meet defined quality control standards. These filters eliminated 7287 probe sets of the 8799 total (82%) from further consideration. Application of judiciously chosen filters is an effective tool for data mining of time series data sets. The remaining data can then be further analyzed by clustering and mathematical modeling techniques.
doi:10.1208/aapsj070117
PMCID: PMC2607485  PMID: 16146338
Data mining; gene arrays; glucocorticoids; mathematical modeling; pharmacogenomics
2.  Corticosteroid-regulated genes in rat kidney: mining time series array data 
Kidney is a major target for adverse effects associated with corticosteroids. A microarray dataset was generated to examine changes in gene expression in rat kidney in response to methylprednisolone. Four control and 48 drug-treated animals were killed at 16 times after drug administration. Kidney RNA was used to query 52 individual Affymetrix chips, generating data for 15,967 different probe sets for each chip. Mining techniques applicable to time series data that identify drug-regulated changes in gene expression were applied. Four sequential filters eliminated probe sets that were not expressed in the tissue, not regulated by drug, or did not meet defined quality control standards. These filters eliminated 14,890 probe sets (94%) from further consideration. Application of judiciously chosen filters is an effective tool for data mining of time series datasets. The remaining data can then be further analyzed by clustering and mathematical modeling. Initial analysis of this filtered dataset identified a group of genes whose pattern of regulation was highly correlated with prototype corticosteroid enhanced genes. Twenty genes in this group, as well as selected genes exhibiting either downregulation or no regulation, were analyzed for 5′ GRE half-sites conserved across species. In general, the results support the hypothesis that the existence of conserved DNA binding sites can serve as an important adjunct to purely analytic approaches to clustering genes into groups with common mechanisms of regulation. This dataset, as well as similar datasets on liver and muscle, are available online in a format amenable to further analysis by others.
doi:10.1152/ajpendo.00196.2005
PMCID: PMC3752664  PMID: 15985454
data mining; gene arrays; glucocorticoids; pharmacogenomics; evolutionary conservation
3.  Temporal profiling of the transcriptional basis for the development of corticosteroid-induced insulin resistance in rat muscle 
The Journal of endocrinology  2005;184(1):219-232.
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.
doi:10.1677/joe.1.05953
PMCID: PMC2574435  PMID: 15642798
4.  Circadian Variations in Liver Gene Expression: Relationships to Drug Actions 
Chronopharmacology is an important but under-explored aspect of therapeutics. Rhythmic variations in biological processes can influence drug action, including pharmacodynamic responses, due to circadian variations in the availability or functioning of drug targets. We hypothesized that global gene expression analysis can be useful in the identification of circadian regulated genes involved in drug action. Circadian variations in gene expression in rat liver were explored using Affymetrix gene arrays. A rich time series involving animals analyzed at 18 time points within the 24 hour cycle was generated. Of the more than 15,000 probe sets on these arrays, 265 exhibited oscillations with a 24 hour frequency. Cluster analysis yielded 5 distinct circadian clusters, with approximately two-thirds of the transcripts reaching maximum expression during the animal’s dark/active period. Of the 265 probe sets, 107 of potential therapeutic importance were identified. The expression levels of clock genes were also investigated in this study. Five clock genes exhibited circadian variation in liver, and data suggest that these genes may also be regulated by corticosteroids.
doi:10.1124/jpet.108.140186
PMCID: PMC2561907  PMID: 18562560
5.  The genomic response of skeletal muscle to methylprednisolone using microarrays: tailoring data mining to the structure of the pharmacogenomic time series 
Pharmacogenomics  2004;5(5):525-552.
High-throughput data collection using gene microarrays has great potential as a method for addressing the pharmacogenomics of complex biological systems. Similarly, mechanism-based pharmacokinetic/pharmacodynamic modeling provides a tool for formulating quantitative testable hypotheses concerning the responses of complex biological systems. As the response of such systems to drugs generally entails cascades of molecular events in time, a time series design provides the best approach to capturing the full scope of drug effects. A major problem in using microarrays for high-throughput data collection is sorting through the massive amount of data in order to identify probe sets and genes of interest. Due to its inherent redundancy, a rich time series containing many time points and multiple samples per time point allows for the use of less stringent criteria of expression, expression change and data quality for initial filtering of unwanted probe sets. The remaining probe sets can then become the focus of more intense scrutiny by other methods, including temporal clustering, functional clustering and pharmacokinetic/pharmacodynamic modeling, which provide additional ways of identifying the probes and genes of pharmacological interest.
doi:10.1517/14622416.5.5.525
PMCID: PMC2607486  PMID: 15212590
corticosteroids; data mining; expression profiling; gene chips; methylprednisolone; microarrays; modeling; pharmacodynamics; skeletal muscle; time series
6.  Extracting Global System Dynamics of Corticosteroid Genomic Effects in Rat Liver 
One of the challenges in constructing biological models involves resolving meaningful data patterns from which the mathematical models will be generated. For models that describe the change of mRNA in response to drug administration, questions exist whether the correct genes have been selected given the myriad transcriptional effects that may occur. Oftentimes, different algorithms will select or cluster different groups of genes from the same data set. A new approach was developed that focuses on identifying the underlying global dynamics of the system instead of selecting individual genes. The procedure was applied to microarray genomic data obtained from rat liver after a large single dose of methylprednisolone in 52 adrenalectomized rats. Twelve clusters of at least 30 genes each were selected, reflecting the major changes over time. This method along with isolating the underlying dynamics of the system also extracts and clusters the genes that make up this global dynamic for further analysis as to the contributions of specific mechanisms affected by the drug.
doi:10.1124/jpet.107.133074
PMCID: PMC3725546  PMID: 18086872
7.  Gene Expression Profiles in Cells of Peripheral Blood Identify New Molecular Markers of Acute Pancreatitis 
Introduction
Blood leukocytes play a major role in mediating local and systemic inflammation during acute pancreatitis. We hypothesize that peripheral blood mononuclear cells (PBMC) in circulation exhibit unique changes in gene expression, and could provide a “reporter” function that reflects the inflammatory response in pancreas of acute pancreatitis.
Methods
To determine specific changes in blood leukocytes during acute pancreatitis, we studied gene transcription profile of in peripheral blood mononuclear cells (PBMC) in a rat model of experimental pancreatitis (sodium taurocholate). Normal rats, saline controls and a model of septic shock were used as a controls. cRNA obtained from PBMC of each group (n = 3) were applied to Affymetrix rat genome DNA Gene Chip Arrays.
Results
From the 8,799 rat genes analyzed, 140 genes showed unique significant changes in their expression in PBMC during the acute phase of pancreatitis, but not in sepsis. Among the 140 genes, 57 were upregulated, while 69 were downregulated. Platelet-derived growth factor receptor, prostaglandin E2 receptor and phospholipase D1 are among the top upregulated genes. Others include genes involved in G protein-coupled receptor and TGF-β-mediated signaling pathways, while genes associated with apoptosis, glucocorticoid receptors and even the cholecystokinin receptor are downregulated.
Conclusions
Microarray analysis in transcriptional profiling of PBMC showed that genes that are uniquely related to molecular and pancreatic function display differential expression in acute pancreatitis. Profiling genes obtained from an easily accessible source during severe pancreatitis may identify surrogate markers for disease severity.
doi:10.1001/archsurg.2007.73
PMCID: PMC2668141  PMID: 18347268
Acute pancreatitis; oligonucleotide microarray; abdominal sepsis; peripheral blood mononuclear cells; PBMC
8.  Pharmacodynamic Modeling of Acute and Chronic Effects of Methylprednisolone on Hepatic Urea Cycle Genes in Rats* 
Corticosteroids (CS) regulate many enzymes at both mRNA and protein levels. This study used microarrays to broadly assess regulation of various genes related to the greater urea cycle and employs pharmacokinetic/pharmacodynamic (PK/PD) modeling to quantitatively analyze and compare the temporal profiles of these genes during acute and chronic exposure to methylprednisolone (MPL). One group of adrenalectomized male Wistar rats received an intravenous bolus dose (50 mg/kg) of MPL, whereas a second group received MPL by a subcutaneous infusion (Alzet osmotic pumps) at a rate of 0.3 mg/kg/hr for seven days. The rats were sacrificed at various time points over 72 hours (acute) or 168 hours (chronic) and livers were harvested. Total RNA was extracted and Affymetrix® gene chips (RG_U34A for acute and RAE 230A for chronic) were used to identify genes regulated by CS. Besides five primary urea cycle enzymes, many other genes related to the urea cycle showed substantial changes in mRNA expression. Some genes that were simply up- or down-regulated after acute MPL showed complex biphasic patterns upon chronic infusion indicating involvement of secondary regulation. For the simplest patterns, indirect response models were used to describe the nuclear steroid-bound receptor mediated increase or decrease in gene transcription (e.g. tyrosine aminotransferase, glucocorticoid receptor). For the biphasic profiles, involvement of a secondary biosignal was assumed (e.g. ornithine decarboxylase, CCAAT/enhancer binding protein) and more complex models were derived. Microarrays were used successfully to explore CS effects on various urea cycle enzyme genes. PD models presented in this report describe testable hypotheses regarding molecular mechanisms and quantitatively characterize the direct or indirect regulation of various genes by CS.
PMCID: PMC2733100  PMID: 19787073
urea cycle; corticosteroids; methylprednisolone; pharmacodynamics; genomics
9.  Characterization of the effect of sample quality on high density oligonucleotide microarray data using progressively degraded rat liver RNA 
BMC Biotechnology  2007;7:57.
Background
The interpretability of microarray data can be affected by sample quality. To systematically explore how RNA quality affects microarray assay performance, a set of rat liver RNA samples with a progressive change in RNA integrity was generated by thawing frozen tissue or by ex vivo incubation of fresh tissue over a time course.
Results
Incubation of tissue at 37°C for several hours had little effect on RNA integrity, but did induce changes in the transcript levels of stress response genes and immune cell markers. In contrast, thawing of tissue led to a rapid loss of RNA integrity. Probe sets identified as most sensitive to RNA degradation tended to be located more than 1000 nucleotides upstream of their transcription termini, similar to the positioning of control probe sets used to assess sample quality on Affymetrix GeneChip® arrays. Samples with RNA integrity numbers less than or equal to 7 showed a significant increase in false positives relative to undegraded liver RNA and a reduction in the detection of true positives among probe sets most sensitive to sample integrity for in silico modeled changes of 1.5-, 2-, and 4-fold.
Conclusion
Although moderate levels of RNA degradation are tolerated by microarrays with 3'-biased probe selection designs, in this study we identify a threshold beyond which decreased specificity and sensitivity can be observed that closely correlates with average target length. These results highlight the value of annotating microarray data with metrics that capture important aspects of sample quality.
doi:10.1186/1472-6750-7-57
PMCID: PMC2082023  PMID: 17854504
10.  Pharmacodynamic/Pharmacogenomic Modeling of Insulin Resistance Genes in Rat Muscle After Methylprednisolone Treatment: Exploring Regulatory Signaling Cascades 
Corticosteroids (CS) effects on insulin resistance related genes in rat skeletal muscle were studied. In our acute study, adrenalectomized (ADX) rats were given single doses of 50 mg/kg methylprednisolone (MPL) intravenously. In our chronic study, ADX rats were implanted with Alzet mini-pumps giving zero-order release rates of 0.3 mg/kg/h MPL and sacrificed at various times up to 7 days. Total RNA was extracted from gastrocnemius muscles and hybridized to Affymetrix GeneChips. Data mining and literature searches identified 6 insulin resistance related genes which exhibited complex regulatory pathways. Insulin receptor substrate-1 (IRS-1), uncoupling protein 3 (UCP3), pyruvate dehydrogenase kinase isoenzyme 4 (PDK4), fatty acid translocase (FAT) and glycerol-3-phosphate acyltransferase (GPAT) dynamic profiles were modeled with mutual effects by calculated nuclear drug-receptor complex (DR(N)) and transcription factors. The oscillatory feature of endothelin-1 (ET-1) expression was depicted by a negative feedback loop. These integrated models provide testable quantitative hypotheses for these regulatory cascades.
PMCID: PMC2733097  PMID: 19787081
corticosteroid; glucocorticoid; microarrays; mathematical modeling; insulin resistance
11.  Salvaging Affymetrix probes after probe-level re-annotation 
BMC Research Notes  2008;1:66.
Background
Affymetrix GeneChips can be re-annotated at the probe-level by breaking up the original probe-sets and recomposing new probe-sets based on up-to-date genomic knowledge, such as available in Entrez Gene. This results in custom Chip Description Files (CDF). Using these custom CDFs improves the quality of the data and thus the results of related gene expression studies. However, 44–71% of the probes on a GeneChip are lost in this re-annotation process. Although generally aimed at less known genes, losing these probes obviously means a substantial loss of expensive experiment data. Biologists are therefore very reluctant to adopt this approach.
Findings
We aimed to re-introduce the non-affected Affymetrix probe-sets after these re-annotation procedures. For this, we developed an algorithm (CDF-Merger) and applied it to standard Affymetrix CDFs and custom Brainarray CDFs to obtain Hybrid CDFs. Thus, salvaging lost Affymetrix probes with our CDF-Merger restored probe content up to 94%. Because the salvaged probes (up to 54% of the probe content on the arrays) represent less-reliable probe-sets, we made the origin of all probe-set definitions traceable, so biologists can choose at any time in their analyses, which subset of probe-sets they want to use.
Conclusion
The availability of up-to-date Hybrid CDFs plus R environment allows for easy implementation of our approach.
doi:10.1186/1756-0500-1-66
PMCID: PMC2547102  PMID: 18710586
12.  Dynamics of Short-Term Gene Expression Profiling in Liver Following Thermal Injury 
The Journal of Surgical Research  2011;176(2):549-558.
Background
Severe trauma, including burns, triggers a systemic response that significantly impacts on the liver, which plays a key role in the metabolic and immune responses aimed at restoring homeostasis. While many of these changes are likely regulated at the gene expression level, there is a need to better understand the dynamics and expression patterns of burn injury-induced genes in order to identify potential regulatory targets in the liver. Herein we characterized the response within the first 24 h in a standard animal model of burn injury using a time series of microarray gene expression data.
Methods
Rats were subjected to a full thickness dorsal scald burn injury covering 20% of their total body surface area while under general anesthesia. Animals were saline resuscitated and sacrificed at defined time points (0, 2, 4, 8, 16, and 24 h). Liver tissues were explanted and analyzed for their gene expression profiles using microarray technology. Sham controls consisted of animals handled similarly but not burned. After identifying differentially expressed probesets between sham and burn conditions over time, the concatenated data sets corresponding to these differentially expressed probesets in burn and sham groups were combined and analyzed using a “consensus clustering” approach.
Results
The clustering method of expression data identified 621 burn-responsive probesets in 4 different co-expressed clusters. Functional characterization revealed that these 4 clusters are mainly associated with pro-inflammatory response, anti-inflammatory response, lipid biosynthesis, and insulin-regulated metabolism. Cluster 1 pro-inflammatory response is rapidly up-regulated (within the first 2 h) following burn injury, while Cluster 2 anti-inflammatory response is activated later on (around 8 h post burn). Cluster 3 lipid biosynthesis is downregulated rapidly following burn, possibly indicating a shift in the utilization of energy sources to produce acute phase proteins which serve the anti-inflammatory response. Cluster 4 insulin-regulated metabolism was down-regulated late in the observation window (around 16 h postburn), which suggests a potential mechanism to explain the onset of hypermetabolism, a delayed but well-known response that is characteristic of severe burns and trauma with potential adverse outcome.
Conclusions
Simultaneous analysis and comparison of gene expression profiles for both burn and sham control groups provided a more accurate estimation of the activation time, expression patterns, and characteristics of a certain burn-induced response based on which the cause-effect relationship among responses were revealed.
doi:10.1016/j.jss.2011.09.052
PMCID: PMC3319855  PMID: 22099593
Burn; gene expression; microarray; inflammation; liver
13.  Effects of High Fat Feeding on Liver Gene Expression in Diabetic Goto-Kakizaki Rats 
Effects of high fat diet (HFD) on obesity and, subsequently, on diabetes are highly variable and modulated by genetics in both humans and rodents. In this report, we characterized the response of Goto-Kakizaki (GK) rats, a spontaneous polygenic model for lean diabetes and healthy Wistar-Kyoto (WKY) controls, to high fat feeding from weaning to 20 weeks of age. Animals fed either normal diet or HFD were sacrificed at 4, 8, 12, 16 and 20 weeks of age and a wide array of physiological measurements were made along with gene expression profiling using Affymetrix gene array chips. Mining of the microarray data identified differentially regulated genes (involved in inflammation, metabolism, transcription regulation, and signaling) in diabetic animals, as well as the response of both strains to HFD. Functional annotation suggested that HFD increased inflammatory differences between the two strains. Chronic inflammation driven by heightened innate immune response was identified to be present in GK animals regardless of diet. In addition, compensatory mechanisms by which WKY animals on HFD resisted the development of diabetes were identified, thus illustrating the complexity of diabetes disease progression.
doi:10.4137/GRSB.S10371
PMCID: PMC3516129  PMID: 23236253
diabetes; high fat diet; gene expression; microarray
14.  Sources of variation in baseline gene expression levels from toxicogenomics study control animals across multiple laboratories 
BMC Genomics  2008;9:285.
Background
The use of gene expression profiling in both clinical and laboratory settings would be enhanced by better characterization of variance due to individual, environmental, and technical factors. Meta-analysis of microarray data from untreated or vehicle-treated animals within the control arm of toxicogenomics studies could yield useful information on baseline fluctuations in gene expression, although control animal data has not been available on a scale and in a form best served for data-mining.
Results
A dataset of control animal microarray expression data was assembled by a working group of the Health and Environmental Sciences Institute's Technical Committee on the Application of Genomics in Mechanism Based Risk Assessment in order to provide a public resource for assessments of variability in baseline gene expression. Data from over 500 Affymetrix microarrays from control rat liver and kidney were collected from 16 different institutions. Thirty-five biological and technical factors were obtained for each animal, describing a wide range of study characteristics, and a subset were evaluated in detail for their contribution to total variability using multivariate statistical and graphical techniques.
Conclusion
The study factors that emerged as key sources of variability included gender, organ section, strain, and fasting state. These and other study factors were identified as key descriptors that should be included in the minimal information about a toxicogenomics study needed for interpretation of results by an independent source. Genes that are the most and least variable, gender-selective, or altered by fasting were also identified and functionally categorized. Better characterization of gene expression variability in control animals will aid in the design of toxicogenomics studies and in the interpretation of their results.
doi:10.1186/1471-2164-9-285
PMCID: PMC2453529  PMID: 18549499
15.  Comprehensive Analysis of PPARα-Dependent Regulation of Hepatic Lipid Metabolism by Expression Profiling 
PPAR Research  2007;2007:26839.
PPARα is a ligand-activated transcription factor involved in the regulation of nutrient metabolism and inflammation. Although much is already known about the function of PPARα in hepatic lipid metabolism, many PPARα-dependent pathways and genes have yet to be discovered. In order to obtain an overview of PPARα-regulated genes relevant to lipid metabolism, and to probe for novel candidate PPARα target genes, livers from several animal studies in which PPARα was activated and/or disabled were analyzed by Affymetrix GeneChips. Numerous novel PPARα-regulated genes relevant to lipid metabolism were identified. Out of this set of genes, eight genes were singled out for study of PPARα-dependent regulation in mouse liver and in mouse, rat, and human primary hepatocytes, including thioredoxin interacting protein (Txnip), electron-transferring-flavoprotein β polypeptide (Etfb), electron-transferring-flavoprotein dehydrogenase (Etfdh), phosphatidylcholine transfer protein (Pctp), endothelial lipase (EL, Lipg), adipose triglyceride lipase (Pnpla2), hormone-sensitive lipase (HSL, Lipe), and monoglyceride lipase (Mgll). Using an in silico screening approach, one or more PPAR response elements (PPREs) were identified in each of these genes. Regulation of Pnpla2, Lipe, and Mgll, which are involved in triglyceride hydrolysis, was studied under conditions of elevated hepatic lipids. In wild-type mice fed a high fat diet, the decrease in hepatic lipids following treatment with the PPARα agonist Wy14643 was paralleled by significant up-regulation of Pnpla2, Lipe, and Mgll, suggesting that induction of triglyceride hydrolysis may contribute to the anti-steatotic role of PPARα. Our study illustrates the power of transcriptional profiling to uncover novel PPARα-regulated genes and pathways in liver.
doi:10.1155/2007/26839
PMCID: PMC2233741  PMID: 18288265
16.  Using gene chips to identify organ-specific, smooth muscle responses to experimental diabetes: potential applications to urological diseases 
BJU international  2007;99(2):418-430.
OBJECTIVE
To identify early diabetes-related alterations in gene expression in bladder and erectile tissue that would provide novel diagnostic and therapeutic treatment targets to prevent, delay or ameliorate the ensuing bladder and erectile dysfunction.
MATERIALS AND METHODS
The RG-U34A rat GeneChip® (Affymetrix Inc., Sunnyvale, CA, USA) oligonucleotide microarray (containing ≈8799 genes) was used to evaluate gene expression in corporal and male bladder tissue excised from rats 1 week after confirmation of a diabetic state, but before demonstrable changes in organ function in vivo. A conservative analytical approach was used to detect alterations in gene expression, and gene ontology (GO) classifications were used to identify biological themes/pathways involved in the aetiology of the organ dysfunction.
RESULTS
In all, 320 and 313 genes were differentially expressed in bladder and corporal tissue, respectively. GO analysis in bladder tissue showed prominent increases in biological pathways involved in cell proliferation, metabolism, actin cytoskeleton and myosin, as well as decreases in cell motility, and regulation of muscle contraction. GO analysis in corpora showed increases in pathways related to ion channel transport and ion channel activity, while there were decreases in collagen I and actin genes.
CONCLUSIONS
The changes in gene expression in these initial experiments are consistent with the pathophysiological characteristics of the bladder and erectile dysfunction seen later in the diabetic disease process. Thus, the observed changes in gene expression might be harbingers or biomarkers of impending organ dysfunction, and could provide useful diagnostic and therapeutic targets for a variety of progressive urological diseases/conditions (i.e. lower urinary tract symptoms related to benign prostatic hyperplasia, erectile dysfunction, etc.).
doi:10.1111/j.1464-410X.2007.06676.x
PMCID: PMC2013735  PMID: 17313427
diabetes mellitus; streptozotocin; erectile dysfunction; bladder dysfunction; microarray analysis; gene chips; genomics
17.  Temporal and functional profile of the transcriptional regulatory network in the early regenerative response to partial hepatectomy in the rat 
BMC Genomics  2008;9:527.
Background
The goal of these studies was to characterize the transcriptional network regulating changes in gene expression in the remnant liver of the rat after 70% partial hepatectomy (PHx) during the early phase response including the transition of hepatocytes from the quiescent (G0) state and the onset of the G1 phase of the cell cycle.
Results
The transcriptome of remnant livers was monitored at 1, 2, 4, and 6 hours after PHx using cDNA microarrays. Differentially regulated genes were grouped into six clusters according their temporal expression profiles. Promoter regions of genes in these clusters were examined for shared transcription factor binding sites (TFBS) by comparing enrichment of each TFBS relative to a reference set using the Promoter Analysis and Interaction Network Toolset (PAINT).
Analysis of the gene expression time series data using ANOVA resulted in a total of 309 genes significantly up- or down-regulated at any of the four time points at a 20% FDR threshold. Sham-operated animals showed no significant differential expression. A subset of the differentially expressed genes was validated using quantitative RT-PCR. Distinct sets of TFBS could be identified that were significantly enriched in each one of the different temporal gene expression clusters. These included binding sites for transcription factors that had previously been recognized as contributing to the onset of regeneration, including NF-κB, C/EBP, HNF-1, CREB, as well as factors, such as ATF, AP-2, LEF-1, GATA and PAX-6, that had not yet been recognized to be involved in this process. A subset of these candidate TFBS was validated by measuring activation of corresponding transcription factors (HNF-1, NK-κB, CREB, C/EBP-α and C/EBP-β, GATA-1, AP-2, PAX-6) in nuclear extracts from the remnant livers.
Conclusion
This analysis revealed multiple candidate transcription factors activated in the remnant livers, some known to be involved in the early phase of liver regeneration, and several not previously identified. The study describes the predominant temporal and functional elements to which these factors contribute and demonstrates the potential of this novel approach to define the functional correlates of the transcriptional regulatory network driving the early response to partial hepatectomy.
doi:10.1186/1471-2164-9-527
PMCID: PMC2613928  PMID: 18990226
18.  A Comparative Study of the Impact of G-Stack Probes on Various Affymetrix GeneChips of Mammalia 
Journal of Nucleic Acids  2010;2010:489736.
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 different 3′ 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.
doi:10.4061/2010/489736
PMCID: PMC2915844  PMID: 20725627
19.  Consistency Analysis of Redundant Probe Sets on Affymetrix Three-Prime Expression Arrays and Applications to Differential mRNA Processing 
PLoS ONE  2009;4(1):e4229.
Affymetrix three-prime expression microarrays contain thousands of redundant probe sets that interrogate different regions of the same gene. Differential expression analysis methods rarely consider probe redundancy, which can lead to inaccurate inference about overall gene expression or cause investigators to overlook potentially valuable information about differential regulation of variant mRNA products. We investigated the behaviour and consistency of redundant probe sets in a publicly-available data set containing samples from mouse brain amygdala and hippocampus and asked how applying filtering methods to the data affected consistency of results obtained from redundant probe sets. A genome-based filter that screens and groups probe sets according to their overlapping genomic alignments significantly improved redundant probe set consistency. Screening based on qualitative Present-Absent calls from MAS5 also improved consistency. However, even after applying these filters, many redundant probe sets showed significant fold-change differences relative to each other, suggesting differential regulation of alternative transcript production. Visual inspection of these loci using an interactive genome visualization tool (igb.bioviz.org) exposed thirty putative examples of differential regulation of alternative splicing or polyadenylation across brain regions in mouse. This work demonstrates how P/A-call and genome-based filtering can improve consistency among redundant probe sets while at the same time exposing possible differential regulation of RNA processing pathways across sample types.
doi:10.1371/journal.pone.0004229
PMCID: PMC2621337  PMID: 19165320
20.  Analysis of probe level patterns in Affymetrix microarray data 
BMC Bioinformatics  2007;8:146.
Background
Microarrays have been used extensively to analyze the expression profiles for thousands of genes in parallel. Most of the widely used methods for analyzing Affymetrix Genechip microarray data, including RMA, GCRMA and Model Based Expression Index (MBEI), summarize probe signal intensity data to generate a single measure of expression for each transcript on the array. In contrast, other methods are applied directly to probe intensities, negating the need for a summarization step.
Results
In this study, we used the Affymetrix rat genome Genechip to explore variability in probe response patterns within transcripts. We considered a number of possible sources of variability in probe sets including probe location within the transcript, middle base pair of the probe sequence, probe overlap, sequence homology and affinity. Although affinity, middle base pair and probe location effects may be seen at the gross array level, these factors only account for a small proportion of the variation observed at the gene level. A BLAST search and the presence of probe by treatment interactions for selected differentially expressed genes showed high sequence homology for many probes to non-target genes.
Conclusion
We suggest that examination and modeling of probe level intensities can be used to guide researchers in refining their conclusions regarding differentially expressed genes. We discuss implications for probe sequence selection for confirmatory analysis using real time PCR.
doi:10.1186/1471-2105-8-146
PMCID: PMC1884176  PMID: 17480226
21.  Bayesian models and meta analysis for multiple tissue gene expression data following corticosteroid administration 
BMC Bioinformatics  2008;9:354.
Background
This paper addresses key biological problems and statistical issues in the analysis of large gene expression data sets that describe systemic temporal response cascades to therapeutic doses in multiple tissues such as liver, skeletal muscle, and kidney from the same animals. Affymetrix time course gene expression data U34A are obtained from three different tissues including kidney, liver and muscle. Our goal is not only to find the concordance of gene in different tissues, identify the common differentially expressed genes over time and also examine the reproducibility of the findings by integrating the results through meta analysis from multiple tissues in order to gain a significant increase in the power of detecting differentially expressed genes over time and to find the differential differences of three tissues responding to the drug.
Results and conclusion
Bayesian categorical model for estimating the proportion of the 'call' are used for pre-screening genes. Hierarchical Bayesian Mixture Model is further developed for the identifications of differentially expressed genes across time and dynamic clusters. Deviance information criterion is applied to determine the number of components for model comparisons and selections. Bayesian mixture model produces the gene-specific posterior probability of differential/non-differential expression and the 95% credible interval, which is the basis for our further Bayesian meta-inference. Meta-analysis is performed in order to identify commonly expressed genes from multiple tissues that may serve as ideal targets for novel treatment strategies and to integrate the results across separate studies. We have found the common expressed genes in the three tissues. However, the up/down/no regulations of these common genes are different at different time points. Moreover, the most differentially expressed genes were found in the liver, then in kidney, and then in muscle.
doi:10.1186/1471-2105-9-354
PMCID: PMC2579308  PMID: 18755028
22.  Comparison of dorsal root ganglion gene expression in rat models of traumatic and HIV-associated neuropathic pain 
To elucidate the mechanisms underlying peripheral neuropathic pain in the context of HIV infection and antiretroviral therapy, we measured gene expression in dorsal root ganglia (DRG) of rats subjected to systemic treatment with the anti-retroviral agent, ddC (Zalcitabine) and concomitant delivery of HIV-gp120 to the rat sciatic nerve. L4 and L5 DRGs were collected at day 14 (time of peak behavioural change) and changes in gene expression were measured using Affymetrix whole genome rat arrays. Conventional analysis of this data set and Gene Set Enrichment Analysis (GSEA) was performed to discover biological processes altered in this model. Transcripts associated with G protein coupled receptor signalling and cell adhesion were enriched in the treated animals, while ribosomal proteins and proteasome pathways were associated with gene down-regulation. To identify genes that are directly relevant to neuropathic mechanical hypersensitivity, as opposed to epiphenomena associated with other aspects of the response to a sciatic nerve lesion, we compared the gp120 + ddC-evoked gene expression with that observed in a model of traumatic neuropathic pain (L5 spinal nerve transection), where hypersensitivity to a static mechanical stimulus is also observed. We identified 39 genes/expressed sequence tags that are differentially expressed in the same direction in both models. Most of these have not previously been implicated in mechanical hypersensitivity and may represent novel targets for therapeutic intervention. As an external control, the RNA expression of three genes was examined by RT-PCR, while the protein levels of two were studied using western blot analysis.
doi:10.1016/j.ejpain.2008.05.011
PMCID: PMC2706986  PMID: 18606552
Neuropathic pain; HIV; Mechanical hypersensitivity; Microarray
23.  Integrating multiple genome annotation databases improves the interpretation of microarray gene expression data 
BMC Genomics  2010;11:50.
Background
The Affymetrix GeneChip is a widely used gene expression profiling platform. Since the chips were originally designed, the genome databases and gene definitions have been considerably updated. Thus, more accurate interpretation of microarray data requires parallel updating of the specificity of GeneChip probes. We propose a new probe remapping protocol, using the zebrafish GeneChips as an example, by removing nonspecific probes, and grouping the probes into transcript level probe sets using an integrated zebrafish genome annotation. This genome annotation is based on combining transcript information from multiple databases. This new remapping protocol, especially the new genome annotation, is shown here to be an important factor in improving the interpretation of gene expression microarray data.
Results
Transcript data from the RefSeq, GenBank and Ensembl databases were downloaded from the UCSC genome browser, and integrated to generate a combined zebrafish genome annotation. Affymetrix probes were filtered and remapped according to the new annotation. The influence of transcript collection and gene definition methods was tested using two microarray data sets. Compared to remapping using a single database, this new remapping protocol results in up to 20% more probes being retained in the remapping, leading to approximately 1,000 more genes being detected. The differentially expressed gene lists are consequently increased by up to 30%. We are also able to detect up to three times more alternative splicing events. A small number of the bioinformatics predictions were confirmed using real-time PCR validation.
Conclusions
By combining gene definitions from multiple databases, it is possible to greatly increase the numbers of genes and splice variants that can be detected in microarray gene expression experiments.
doi:10.1186/1471-2164-11-50
PMCID: PMC2827411  PMID: 20089164
24.  Exploring drug action on Mycobacterium tuberculosis using affymetrix oligonucleotide genechips 
Summary
DNA microarrays have rapidly emerged as an important tool for Mycobacterium tuberculosis research. While the microarray approach has generated valuable information, a recent survey has found a lack of correlation among the microarray data produced by different laboratories on related issues, raising a concern about the credibility of research findings. The Affymetrix oligonucleotide array has been shown to be more reliable for interrogating changes in gene expression than other platforms. However, this type of array system has not been applied to the pharmacogenomic study of M. tuberculosis. The goal here was to explore the strength of the Affymetrix array system for monitoring drug-induced gene expression in M. tuberculosis, compare with other related studies, and conduct cross-platform analysis. The genome-wide gene expression profiles of M. tuberculosis in response to drug treatments including INH (isoniazid) and ethionamide were obtained using the Affymetrix array system. Up-regulated or down-regulated genes were identified through bioinformatic analysis of the microarray data derived from the hybridization of RNA samples and gene probes. Based on the Affymetrix system, our method identified all drug-induced genes reported in the original reference work as well as some other genes that have not been recognized previously under the same drug treatment. For instance, the Affymetrix system revealed that Rv2524c (fas) was induced by both INH and ethionamide under the given levels of concentration, as suggested by most of the probe sets implementing this gene sequence. This finding is contradictory to previous observations that the expression of fas is not changed by INH treatment. This example illustrates that the determination of expression change for certain genes is probe-dependent, and the appropriate use of multiple probe-set representation is an advantage with the Affymetrix system. Our data also suggest that whereas the up-regulated gene expression pattern reflects the drug’s mode of action, the down-regulated pattern is largely non-specific. According to our analysis, the Affymetrix array system is a reliable tool for studying the pharmacogenomics of M. tuberculosis and lends itself well in the research and development of anti-TB drugs.
doi:10.1016/j.tube.2005.07.004
PMCID: PMC1557687  PMID: 16246625
Tuberculosis; Drug; Microarray; Genome
25.  The effect of oligonucleotide microarray data pre-processing on the analysis of patient-cohort studies 
BMC Bioinformatics  2006;7:105.
Background
Intensity values measured by Affymetrix microarrays have to be both normalized, to be able to compare different microarrays by removing non-biological variation, and summarized, generating the final probe set expression values. Various pre-processing techniques, such as dChip, GCRMA, RMA and MAS have been developed for this purpose. This study assesses the effect of applying different pre-processing methods on the results of analyses of large Affymetrix datasets. By focusing on practical applications of microarray-based research, this study provides insight into the relevance of pre-processing procedures to biology-oriented researchers.
Results
Using two publicly available datasets, i.e., gene-expression data of 285 patients with Acute Myeloid Leukemia (AML, Affymetrix HG-U133A GeneChip) and 42 samples of tumor tissue of the embryonal central nervous system (CNS, Affymetrix HuGeneFL GeneChip), we tested the effect of the four pre-processing strategies mentioned above, on (1) expression level measurements, (2) detection of differential expression, (3) cluster analysis and (4) classification of samples. In most cases, the effect of pre-processing is relatively small compared to other choices made in an analysis for the AML dataset, but has a more profound effect on the outcome of the CNS dataset. Analyses on individual probe sets, such as testing for differential expression, are affected most; supervised, multivariate analyses such as classification are far less sensitive to pre-processing.
Conclusion
Using two experimental datasets, we show that the choice of pre-processing method is of relatively minor influence on the final analysis outcome of large microarray studies whereas it can have important effects on the results of a smaller study. The data source (platform, tissue homogeneity, RNA quality) is potentially of bigger importance than the choice of pre-processing method.
doi:10.1186/1471-2105-7-105
PMCID: PMC1481623  PMID: 16512908

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