Illumina BeadArray Research Articles

Differential Gene Expression Profile of the Mouse Oviduct During the Estrous Cycle.

The mammalian oviduct provides suitable environment for gamete's transport, interaction, fertilization and early embryo development. Gamete's arrival in the oviduct may cause distinct alterations in the oviductal gene expression. So far, there are only few studies providing the limited information regarding the oviductal gene expression profile. It is thus still not enough for better understanding of oviductal physiology. In this study, we attempted to reveal the profile of the oviductal transcriptome at each stage of estrous cycle by microarray analyses. The oviductal tissue was separately collected at four stage of the estrous cycle, i.e., proestrus, estrus, metestrus, and diestrus. The estrous cycle was staged by examining vaginal smears. One microgram of total RNA was transcribed into cDNA, reversed transcribed via in vitro transcription, and labeled with biotin. The bead-based microarray (Illumina BeadArray) system was used to profile the oviductal transcriptome at each stage of the estrous cycle. Microarray data were validated by quantitative reverse transcription-PCR (real-time PCR) measurements. Cluster analysis of microarray data was performed using Genesis software. Ingenuity Pathways Analysis (IPA) software was used for finding function and pathway for specific biological states. Our results showed that differentially expressed genes, 199, 131, and 13, genes with a 1.5-fold difference were observed in the oviduct at proestrus, estrus, and metestrus, respectively, as compared individually with the diestrus stage. Ten randomly selected genes were selected and demonstrated to have the same expression trend between the microarray and real-time PCR analysis. These differentially expressed genes could be classified into 5 clusters according the expression pattern. Gene interaction analysis using IPA tool revealed at least 10 associated network functions. Among them, lipid metabolism, development and function of reproductive system, and endocrine system are the most associated network functions. This is the first study to provide a comprehensive gene expression profile of oviduct at the physiological estrus cycle. It may also provide information for our better understanding the complexity of the oviductal biology in creating a hospital environment for gametes interaction. (poster)

Primary Hyperparathyroidism Influences the Expression of Inflammatory and Metabolic Genes in Adipose Tissue

BackgroundPrimary hyperparathyroidism (PHPT) is characterised by increased production of parathyroid hormone (PTH) resulting in elevated serum calcium levels. The influence on bone metabolism with altered bone resorption is the most studied clinical condition in PHPT. In addition to this, patients with PHPT are at increased risk of non-skeletal diseases, such as impaired insulin sensitivity, arterial hypertension and increased risk of death by cardiovascular diseases (CVD), possibly mediated by a chronic low-grade inflammation. The aim of this study was to investigate whether adipose tissue reflects the low-grade inflammation observed in PHPT patients.Methodology/Principal FindingsSubcutaneous fat tissue from the neck was sampled from 16 non-obese patients with PHPT and from 16 patients operated for benign thyroid diseases, serving as weight-matched controls. RNA was extracted and global gene expression was analysed with Illumina BeadArray Technology. We found 608 differentially expressed genes (q-value<0.05), of which 347 were up-regulated and 261 were down-regulated. Gene ontology analysis showed that PHPT patients expressed increased levels of genes involved in immunity and defense (e.g. matrix metallopeptidase 9, S100 calcium binding protein A8 and A9, CD14, folate receptor 2), and reduced levels of genes involved in metabolic processes. Analysis of transcription factor binding sites present in the differentially expressed genes corroborated the up-regulation of inflammatory processes.Conclusions/SignificanceOur findings demonstrate that PHPT strongly influences gene regulation in fat tissue, which may result in altered adipose tissue function and release of pathogenic factors that increase the risk of CVD.

Role of Pyk2 in cardiac arrhythmogenesis

Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor protein kinase regulated by intracellular Ca(2+), CaMK, and PKC and can be activated by different stress signals involved in heart failure. However, Pyk2 has not been investigated in the human heart, and the functional role of Pyk2 signaling at the whole heart level has not been elucidated. We hypothesize that Ca(2+)-dependent activation of Pyk2 is involved in cardiac electrophysiology. We examined the expression of Pyk2 in nonfailing versus ischemic and nonischemic failing human hearts (n = 6 hearts/group). To investigate Pyk2 function, we optically mapped perfused hearts from wild-type (WT; n = 7) and knockout (Pyk2(-/-); n = 8) mice during autonomic stimulation. Experiments were done in control mice and after 1 wk of transverse aortic constriction. We used the Illumina beadarray approach for transcriptional profiling of WT and Pyk2(-/-) mouse ventricles. Western blot analysis revealed a doubling of Pyk2 activation in nonischemic failing versus nonfailing human hearts. In mouse hearts, we observed a much higher probability of ventricular tachyarrhythmia during ACh perfusion in Pyk2(-/-) versus WT mice. Parasympathetic stimulation resulted in a dose-dependent decrease of atrial action potential duration (APD) in both WT and Pyk2(-/-) mice, whereas in ventricles it induced APD shortening in Pyk2(-/-) mice but not in WT mice. Deficiency of Pyk2 abolished ACh-induced prolongation of atrioventricular delay in Pyk2(-/-) mouse hearts but did not affect heart rate. Lower mRNA and protein levels of sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 and higher mRNA levels of Na(+)/Ca(2+) exchanger 1 were detected in Pyk2(-/-) hearts compared with WT hearts. The transverse aortic constriction protocol did not change the phenotype. In conclusion, our results indicate a protective role of Pyk2 with respect to ventricular tachyarrhythmia during parasympathetic stimulation by regulation of gene expression related to Ca(2+) handling. We hypothesize that activation of Pyk2 in the human heart during heart failure may contribute to protection against arrhythmia.

Relevance of germ-line genetic variations for treatment response in pediatric osteosarcoma patients.

9518 Background: Osteosarcoma comprises 5% of all pediatric malignancies and only 50% of the patients will survive. A poor response to chemotherapy is the dominant risk factor for poor survival. Pharmacogenetic research can help to understand the mechanism of drug response in these patients and offers the possibility to optimise treatment and improve outcome. Therefore, we applied a pathway-based approach to evaluate the cumulative effect of genes involved in the transport and metabolism of cisplatin and doxorubicin in relationship to clinical outcome of patients with osteosarcoma. Methods: We used a two stage approach to comprehensively evaluate the role of polymorphisms in genes involved in the cisplatinum and doxorubicin pathway. For the first stage we included 107 patients with osteosarcoma, 31 with good histologic respons (huvos III/IV) following neoadjuvant treatment and 76 patients with bad histologic respons (huvos I/II). A total of 54 candidate genes were selected by a pathway-driven approach based on literature, using databases from NCBI (PubMed, Gene, and SNP) and the Pharmacogenomics Knowledge Base. In order to comprehensively assess common genetic variation in the genes selected, a linkage disequilibrium (LD) based tag-SNP strategy was employed. A final set of 384 SNPs were typed using Illumina Beadarray platform. Results: Of the 384 SNPs genotyped, 353 SNPs passed our initial quality control criteria, and of these, two were excluded by PLINK default thresholds. Another 20 SNPs of the remaining 351 SNPs failed to meet Hardy-Weinberg equilibrium (HWE) criteria (PHWE<0.05). Of the remaining 331 SNPs analyzed in the discovery stage, a total of 24 SNPs with PTrend<0.05 in the cisplatina doxorubicine pathway were associated with histological response. In addition, a clear associations betweenthe development of acute cardiotoxicity and the presence of SNPs in the doxorubicine pathway were observed. Conclusions: This pharmacogenetic results might be useful to stratify treatment of patients immediately after diagnosis and offer the possibility to improve treatment and disease free survival of pediatric patients with osteosarcoma.

Genome-Wide DNA Methylation Profiles in Noncancerous Gastric Mucosae with Regard to Helicobacter pylori Infection and the Presence of Gastric Cancer

To determine genome-wide DNA methylation profiles induced by Helicobacter pylori (H. pylori) infection and to identify methylation markers in H. pylori-induced gastric carcinogenesis. Gastric mucosae obtained from controls (n = 20) and patients with gastric cancer (n = 28) were included. A wide panel of CpG sites in cancer-related genes (1505 CpG sites in 807 genes) was analyzed using Illumina bead array technology. Validation of the results of Illumina bead array technique was performed using methylation-specific PCR method for four genes (MOS, DCC, CRK, and PTPN6). The Illumina bead array showed that a total of 359 CpG sites (269 genes) were identified as differentially methylated by H. pylori infection (p < .0001). The correlation between methylation-specific PCR and bead array analysis was significant (p < .0001, Spearman coefficient = 0.5054). Methylation profiles in noncancerous gastric mucosae of the patients with gastric cancer showed quite distinct patterns according to the presence or absence of the current H. pylori infection; however, 10 CpG sites were identified to be hypermethylated and three hypomethylated in association with the presence of gastric cancer regardless of H. pylori infection (p < .01). Genome-wide methylation profiles showed a number of genes differentially methylated by H. pylori infection. Methylation profiles in noncancerous gastric mucosae from the patients with gastric cancer can be affected by H. pylori-induced gastritis. Differentially methylated CpG sites in this study needs to be validated in a larger population using quantitative methylation-specific PCR method.

Abstract 5350: Distinction of childhood soft tissue sarcomas with high metastatic potential by miRNAs

Abstract Background: Advanced stage alveolar (RMA) and embryonal rhabdomyosarcoma (RME) as well as malignant rhabdoid tumor (MRT) have a poor prognosis. Novel treatment approaches based on an improved understanding of metastatic invasion are required. Aberrant microRNA (miRNA) expression is found in cancer progression and metastatic invasion and may help to identify novel targets. The aim of this study was to identify specific miRNA expression patterns in pediatric soft tissue sarcomas. Methods: We analyzed the expression of 743 miRNAs in 5 RMA, RME, and MRT formalin fixed tissue samples and in the rhabdomyosarcoma (RMS) cell lines Rh30 and RD using an Illumina BeadArray. Differentially expressed miRNAs between RMS and MRT specimens were identified on the basis of the cubic spline algorithm using BeadStudio Software. Selected target miRNAs were transfected with mimic or inhibitor oligonucleotides. Functional analysis was monitored by western blotting, flow cytometry, and migration assays. Results: Principal component analysis as well as Spearman's rank correlation on the set of differentially expressed miRNAs showed tissue-specific clustering. A review of the literature revealed 23 miRNAs promoting and 46 miRNAs suppressing metastatic invasion. Eight miRNAs regulating migration in both directions were also described. In our study, metastatic invasion associated miRNAs miR-9* and miR-221 were overexpressed in RMS tissue samples compared to MRT. miR-200c, which inhibits metastatic invasion, was lower expressed in RMS than in MRT. Transient transfection of RMS cells with a miR-200c mimic resulted neither in a reduction of the direct target ZEB1 nor in an increase of the ZEB1-repressed ectopic E-cadherin expression. Migration of RMS cells after induction of miR-200c was not decreased. Furthermore, inhibition of miR-9* and miR-221 in RMS cell lines did not influence migration characteristics. Conclusions: Pediatric RMS and MRT can be distinguished by their specific miRNA expression pattern. These might also help for an optimized risk stratification of these tumors. A modulation of the metastatic invasion was not possible by the investigated miRNAs. Therefore, regulation of migration seems to be controlled by multiple miRNAs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5350. doi:10.1158/1538-7445.AM2011-5350

Abstract 4657: Identification and validation of genetic determinants of DNA repair phenotype in genome-wide association study of lung cancer

Abstract DNA repair is essential in protecting the integrity of the genome. There is considerable inter-individual variation in DNA repair capacity (DRC). We have reported that cellular DRC (measured in lymphocytes using the host-cell reactivation assay with a reporter gene chloramphenicol acetyltransferase [CAT] damaged by an activated tobacco carcinogen, benzo(a)pyrene diol epoxide [BPDE]) modulates susceptibility to lung cancer. To identify genetic polymorphisms predictive of the repair phenotype, we used phenotype and genotype data in a population of 1,474 European ancestry individuals from a Texas lung cancer genome-wide association case-control study using Illumina 317K platform (317498 SNPs). This discovery phase was followed by replication testing of the top 20 SNPs in an independent set of an additional 1,364 European ancestry cases and controls, using Illumina Bead Array Platform. In addition, we performed a replication analysis of all SNPs with P &amp;lt; 0.01 in 145 DNA repair-related genes in BER, NER, MMR, DSBR pathways from the discovery set, and applying a conservative Bonferroni-corrected P -value of 2.37E-5 for the joint P -value, based on the total number of analyzed SNPs in DNA repair genes. The DRC data were divided into quartiles by assigning the top quarter of participants (with highest values of DRC) as the DNA repair proficient group and the bottom quarter of participants with lowest values of DRC as the suboptimal DRC group to enhance the contrast. We used a generalized linear model with SNPs as predictors and DRC as the outcome. The covariates age, gender, smoking status, duration of smoking, number of cigarettes per day, DRC assay-related variables including the sample storage time, baseline reporter gene CAT expression level, blastogenic rate, and the case-control status of the study participants were adjusted in the model. We identified several suggestive loci contributing to the DRC phenotype: rs9390123 in PHACTR2 (phosphatase and actin regulator 2, P = 4.78E-05 in the discovery set and P = 1.08E-05 in the joint analysis); rs4756897 in USH1C (Usher syndrome 1C, P = 5.40E-05 in the discovery set and 8.71E-06 in the joint analysis); rs6785626 in CNTN4 (contactin 4, P = 0.000451 in the discovery set and P = 1.55E-05 in the joint analysis); rs13181 in ERCC2 (excision repair cross-complementing rodent repair deficiency-complementation group 2, joint P = 2.09E-05); rs7443927 in DUSP1 (dual specificity phosphatase 1, joint P =5.04E-05) and rs10878361 in HMGA2 (high mobility group AT-hook 2, joint P =2.54E-4). In conclusion, we identified and replicated a number of SNPs associated with DRC. These genetic determinants of DNA repair phenotype are easier and cheaper to measure, and subject to less misclassification than the phenotype and may be effective biomarkers for risk assessment. (This study was supported by NIH grants CA127219, CA055769, ES011740, CA131274 and CA016672) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4657. doi:10.1158/1538-7445.AM2011-4657

Presence of evolutionary conserved gene expression signatures during progression of Barrett's esophagus to esophageal adenocarcinoma in human and rat

Esophageal adenocarcinoma (EAC) in Barrett's esophagus (BE) patients is thought to arise from a sequence of growth‐promoting genetic alterations due to chronic esophagitis. However the molecular events underlying the pathogenesis of BE and EAC remain largely unclear. To define the biological mechanisms of EAC development, we performed a cross‐species comparison of gene expression data from human and rat tissues, seeking the evolutionarily conserved gene expression patterns during tumor progression across both species. We used a non‐carcinogenic rat model that closely resembling the natural history of EAC development in human. Using Illumina beadarray platform, we first generated gene expression data from human tissues 28 NE, 72 BE, and 75 EAC as well as from rat tissues 15 NE, 18 BE, and 30 EAC. Statistical analyses on human and rat gene expression data revealed gene expression patterns significantly associated with progression of disease (NE to BE and BE to EAC). Using orthologous genes presented in both microarray platforms, we identified 397 genes are commonly altered as NE progresses to BE and 205 genes including SPP1 were altered as BE progresses to EAC in both species. Systems‐level characterization of gene expression data from human and rodent could become reliable biomarkers for early detection or therapeutic targets for treatment of EAC patients.

Microarray assessment of the influence of the conceptus on gene expression in the mouse uterus during decidualization.

During pregnancy in several species including humans and rodents, the endometrium undergoes decidualization. This process of differentiation from endometrial to decidual tissue occurs only after the onset of implantation in mice. It can also be artificially induced causing the formation of deciduomal tissue. The purpose of this study was to compare the gene expression profile of the developing decidua in pregnant mice with the deciduoma formed after artificial induction in an effort to identify conceptus-influenced changes in uterine gene expression during decidualization. We induced decidualization artificially by transferring blastocyst-sized ConA-coated agarose beads into the uterus on day 2.5 of pseudopregnancy. Recently published work has found this model to be more 'physiological' than other methods. Total RNA was isolated from blastocyst and bead-induced 'implantation' sites of the uteri of day 7.5 pregnant (decidua) and pseudopregnant (deciduoma) mice respectively. This RNA was then used for microarray analysis using Mouse Illumina BeadArray chips. This analysis revealed potential differential mRNA levels of only 45 genes between the decidua and bead-induced deciduoma tissues. We confirmed the differential mRNA levels of 31 of these genes using quantitative RT-PCR. Finally, the level and localization of some of the mRNAs for select genes (Aldh3a1, Bcmo1, Guca2b, and Inhbb) identified by our microarray analysis were examined in more detail. This study provides the identity of a small set of genes whose expression in the uterus during decidualization may be influenced by molecular signals from the conceptus.

Genome-wide analysis of Ollier disease: Is it all in the genes?

BackgroundOllier disease is a rare, non-hereditary disorder which is characterized by the presence of multiple enchondromas (ECs), benign cartilaginous neoplasms arising within the medulla of the bone, with an asymmetric distribution. The risk of malignant transformation towards central chondrosarcoma (CS) is increased up to 35%. The aetiology of Ollier disease is unknown.MethodsWe undertook genome-wide copy number and loss of heterozygosity (LOH) analysis using Affymetrix SNP 6.0 array on 37 tumours of 28 Ollier patients in combination with expression array using Illumina BeadArray v3.0 for 7 ECs of 6 patients.ResultsNon-recurrent EC specific copy number alterations were found at FAM86D, PRKG1 and ANKS1B. LOH with copy number loss of chromosome 6 was found in two ECs from two unrelated Ollier patients. One of these patients also had LOH at chromosome 3. However, no common genomic alterations were found for all ECs. Using an integration approach of SNP and expression array we identified loss as well as down regulation of POU5F1 and gain as well as up regulation of NIPBL. None of these candidate regions were affected in more than two Ollier patients suggesting these changes to be random secondary events in EC development. An increased number of genetic alterations and LOH were found in Ollier CS which mainly involves chromosomes 9p, 6q, 5q and 3p.ConclusionsWe present the first genome-wide analysis of the largest international series of Ollier ECs and CS reported so far and demonstrate that copy number alterations and LOH are rare and non-recurrent in Ollier ECs while secondary CS are genetically unstable. One could predict that instead small deletions, point mutations or epigenetic mechanisms play a role in the origin of ECs of Ollier disease.

CNV-WebStore: Online CNV Analysis, Storage and Interpretation

BackgroundMicroarray technology allows the analysis of genomic aberrations at an ever increasing resolution, making functional interpretation of these vast amounts of data the main bottleneck in routine implementation of high resolution array platforms, and emphasising the need for a centralised and easy to use CNV data management and interpretation system.ResultsWe present CNV-WebStore, an online platform to streamline the processing and downstream interpretation of microarray data in a clinical context, tailored towards but not limited to the Illumina BeadArray platform. Provided analysis tools include CNV analsyis, parent of origin and uniparental disomy detection. Interpretation tools include data visualisation, gene prioritisation, automated PubMed searching, linking data to several genome browsers and annotation of CNVs based on several public databases. Finally a module is provided for uniform reporting of results.ConclusionCNV-WebStore is able to present copy number data in an intuitive way to both lab technicians and clinicians, making it a useful tool in daily clinical practice.

Genome-Wide Association Studies Reveal Genetic Variants in CTNND2 for High Myopia in Singapore Chinese

To determine susceptibility genes for high myopia in Singaporean Chinese. A meta-analysis of 2 genome-wide association (GWA) datasets in Chinese and a follow-up replication cohort in Japanese. Two independent datasets of Singaporean Chinese individuals aged 10 to 12 years (Singapore Cohort Study of the Risk factors for Myopia [SCORM]: cases = 65, controls = 238) and more than 21 years (Singapore Prospective Study Program [SP2]: cases = 222, controls = 435) for GWA studies, and a Japanese dataset aged more than 20 years (cases = 959, controls = 2128) for replication. Genomic DNA samples from SCORM and SP2 were genotyped using various Illumina Beadarray platforms (>HumanHap 500). Single-locus association tests were conducted for each dataset with meta-analysis using pooled z-scores. The top-ranked genetic markers were examined for replication in the Japanese dataset. Fisher P was calculated for the combined analysis of all 3 cohorts. High myopia, defined by spherical equivalent (SE) ≤ -6.00 diopters (D); controls defined by SE between -0.50 and +1.00 D. Two SNPs (rs12716080 and rs6885224) in the gene CTNND2 on chromosome 5p15 ranked top in the meta-analysis of our Chinese datasets (meta P = 1.14 × 10(-5) and meta P = 1.51 × 10(-5), respectively) with strong supporting evidence in each individual dataset analysis (max P = 1.85 × 10(-4) in SCORM: max P = 8.8 × 10(-3) in SP2). Evidence of replication was observed in the Japanese dataset for rs6885224 (P = 0.035, meta P of 3 datasets: 7.84 × 10(-6)). This study identified a strong association of CTNND2 for high myopia in Asian datasets. The CTNND2 gene maps to a known high myopia linkage region on chromosome 5p15.

한우의 유전체 표지인자 활용 개체 혈연관계 추정

한우의 유전체 전장의 정보를 Illumina BeadArray<TEX>$^{TM}$</TEX> Bovine SNP50 assay를 이용하여 단일염기다형 현상을 조사한 결과, 유전적 다양성을 보이는 좌위가 약 32,567 좌위 이상에서 다양성을 보이고 있었으며 약 5,554 좌위에서 다양성이 조사되지 않았다. 이는 조사된 자료의 가계집단의 수가 크게 제한되었기 때문에 기인될 수 있으며 또 다른 원인으로는 한우 종축집단의 크기가 작을 수 있다는 현상을 반증한다고 사료된다. 유전분석의 기초가 되는 혈통기록에 의한 개체간 혈연관계를 유전체 정보에 의한 혈연관계와 비교하여 본 결과, 유전체 정보에 의한 혈연관계의 크기가 혈통기록에 의한 혈연관계보다 좀 더 정확하게 추정될 수 있다는 장점이 있으며 혈통기록상의 오류로 그릇된 혈연관계의 크기를 유전체 정보를 통하여 보완할 수 있다는 장점이 있다. 이러한 장점을 활용하면 유전체정보를 이용한 유전능력 평가의 정확성을 크게 향상시킬 수 있을 것으로 사료되었다. The emergence of next-generation sequencing technologies has lead to application of new computational and statistical methodologies that allow incorporating genetic information from entire genomes of many individuals composing the population. For example, using single-nucleotide polymorphisms (SNP) obtained from whole genome amplification platforms such as the Ilummina BovineSNP50 chip, many researchers are actively engaged in the genetic evaluation of cattle livestock using whole genome relationship analyses. In this study, we estimated the genomic relationship matrix (GRM) and compared it with one computed using a pedigree relationship matrix (PRM) using a population of Hanwoo. This project is a preliminary study that will eventually include future work on genomic selection and prediction. Data used in this study were obtained from 187 blood samples consisting of the progeny of 20 young bulls collected after parentage testing from the Hanwoo improvement center, National Agriculture Cooperative Federation as well as 103 blood samples from the progeny of 12 proven bulls collected from farms around the Kyong-buk area in South Korea. The data set was divided into two cases for analysis. In the first case missing genotypes were included. In the second case missing genotypes were excluded. The effect of missing genotypes on the accuracy of genomic relationship estimation was investigated. Estimation of relationships using genomic information was also carried out chromosome by chromosome for whole genomic SNP markers based on the regression method using allele frequencies across loci. The average correlation coefficient and standard deviation between relationships using pedigree information and chromosomal genomic information using data which was verified using a parentage test andeliminated missing genotypes was <TEX>$0.81{\pm}0.04$</TEX> and their correlation coefficient when using whole genomic information was 0.98, which was higher. Variation in relationships between non-inbred half sibs was <TEX>$0.22{\pm}0.17$</TEX> on chromosomal and <TEX>$0.22{\pm}0.04$</TEX> on whole genomic SNP markers. The variations were larger and unusual values were observed when non-parentage test data were included. So, relationship matrix by genomic information can be useful for genetic evaluation of animal breeding.

The cost of reducing starting RNA quantity for Illumina BeadArrays: A bead-level dilution experiment

BackgroundThe demands of microarray expression technologies for quantities of RNA place a limit on the questions they can address. As a consequence, the RNA requirements have reduced over time as technologies have improved. In this paper we investigate the costs of reducing the starting quantity of RNA for the Illumina BeadArray platform. This we do via a dilution data set generated from two reference RNA sources that have become the standard for investigations into microarray and sequencing technologies.ResultsWe find that the starting quantity of RNA has an effect on observed intensities despite the fact that the quantity of cRNA being hybridized remains constant. We see a loss of sensitivity when using lower quantities of RNA, but no great rise in the false positive rate. Even with 10 ng of starting RNA, the positive results are reliable although many differentially expressed genes are missed. We see that there is some scope for combining data from samples that have contributed differing quantities of RNA, but note also that sample sizes should increase to compensate for the loss of signal-to-noise when using low quantities of starting RNA.ConclusionsThe BeadArray platform maintains a low false discovery rate even when small amounts of starting RNA are used. In contrast, the sensitivity of the platform drops off noticeably over the same range. Thus, those conducting experiments should not opt for low quantities of starting RNA without consideration of the costs of doing so. The implications for experimental design, and the integration of data from different starting quantities, are complex.

A statistical framework for Illumina DNA methylation arrays

The Illumina BeadArray is a popular platform for profiling DNA methylation, an important epigenetic event associated with gene silencing and chromosomal instability. However, current approaches rely on an arbitrary detection P-value cutoff for excluding probes and samples from subsequent analysis as a quality control step, which results in missing observations and information loss. It is desirable to have an approach that incorporates the whole data, but accounts for the different quality of individual observations. We first investigate and propose a statistical framework for removing the source of biases in Illumina Methylation BeadArray based on several positive control samples. We then introduce a weighted model-based clustering called LumiWCluster for Illumina BeadArray that weights each observation according to the detection P-values systematically and avoids discarding subsets of the data. LumiWCluster allows for discovery of distinct methylation patterns and automatic selection of informative CpG loci. We demonstrate the advantages of LumiWCluster on two publicly available Illumina GoldenGate Methylation datasets (ovarian cancer and hepatocellular carcinoma). R package LumiWCluster can be downloaded from http://www.unc.edu/∼pfkuan/LumiWCluster.

HYPERTENSIVE RENAL DAMAGE IS NOT AGEING: EVIDENCE FROM GLOBAL GENE EXPRESSION IN RAT RENAL CORTEX: 7D.02

Objectives: Separating the effects of chronic hypertension on renal structure and function from those of ageing is difficult. The present investigation addresses this question by comparing three microarray studies of mRNA expression in the kidney cortex. Design: 60 week old spontaneously hypertensive rats (SHR) were compared with normotensive Wistar Kyoto rats; rats with renal hypertension for 24 weeks with age matched controls and 4 weeks hypertension (2K1C); and 90 week old normotensive rats with 10 week old rats (old rats). Methods: Global gene expression was assayed using Illumina Bead-array cDNA microarrays. The experiments were analysed using R, Bioconductor and Beadarray. Each experiment was analysed separately and the resultant gene-lists compared. Interstitial fibrosis was investigated using sirius red in histological sections. Results: Using the gene ontology enrichment analysis software toolkit (GOEAST) the enrichment of up and down-regulated genes were investigated. In both SHR and 2K1C showed an enrichment of up-regulated genes relating to the extracellular matrix (ECM). This corresponds to the well known hallmarks of chronic renal damage; tubular atropht and interstitial fibrosis. Interestingly, the ECM was enriched with down-regulated genes in old rats. In SHR the up-regulated ECM genes included fibronectin and decorin, as well as several types of fibrillar collagen: Col1a1, Col1a2 and Col3a1. In addition, the matrix metalloproteases MMP14 and MMP9, the tissue inhibitor of metalloproteases, TIMP1 and TIMP2, and the procollagen peptidase ADAMTS1 were up-regulated. In 2K1C, ADAMTS1 was up-regulated, together with matrix Gla protein, decorin, Lgals1, Lgals3, MMP12, and TIMP1 all proteins involved in collagen deposition and turnover. In contrast, old rats showed downregulation of several forms of collagen: Col1a1, Col4a1, Col1a2, Col3a1 and Col6a3. In addition, the expressions of MMP9 and Sparc were decreased. Histologically SHR and 2K1C showed focal lesions with tubular damage and interstitial fibrosis, while old rats showed diffuse increase of interstitial collagen. Conclusion: The genetic regulation of ECM differs radically between normal ageing and chronic hypertensive renal damage.

A Truncating Mutation in SERPINB6 Is Associated with Autosomal-Recessive Nonsyndromic Sensorineural Hearing Loss

More than 270 million people worldwide have hearing loss that affects normal communication. Although astonishing progress has been made in the identification of more than 50 genes for deafness during the past decade, the majority of deafness genes are yet to be identified. In this study, we mapped a previously unknown autosomal-recessive nonsyndromic sensorineural hearing loss locus (DFNB91) to chromosome 6p25 in a consanguineous Turkish family. The degree of hearing loss was moderate to severe in affected individuals. We subsequently identified a nonsense mutation (p.E245X) in SERPINB6, which is located within the linkage interval for DFNB91 and encodes for an intracellular protease inhibitor. The p.E245X mutation cosegregated in the family as a completely penetrant autosomal-recessive trait and was absent in 300 Turkish controls. The mRNA expression of SERPINB6 was reduced and production of protein was absent in the peripheral leukocytes of homozygotes, suggesting that the hearing loss is due to loss of function of SERPINB6. We also demonstrated that SERPINB6 was expressed primarily in the inner ear hair cells. We propose that SERPINB6 plays an important role in the inner ear in the protection against leakage of lysosomal content during stress and that loss of this protection results in cell death and sensorineural hearing loss.

Identification and correction of previously unreported spatial phenomena using raw Illumina BeadArray data

BackgroundA key stage for all microarray analyses is the extraction of feature-intensities from an image. If this step goes wrong, then subsequent preprocessing and processing stages will stand little chance of rectifying the matter. Illumina employ random construction of their BeadArrays, making feature-intensity extraction even more important for the Illumina platform than for other technologies. In this paper we show that using raw Illumina data it is possible to identify, control, and perhaps correct for a range of spatial-related phenomena that affect feature-intensity extraction.ResultsWe note that feature intensities can be unnaturally high when in the proximity of a number of phenomena relating either to the images themselves or to the layout of the beads on an array. Additionally we note that beads neighbour beads of the same type more often than one might expect, which may cause concern in some models of hybridization. We highlight issues in the identification of a bead's location, and in particular how this both affects and is affected by its intensity. Finally we show that beads can be wrongly identified in the image on either a local or array-wide scale, with obvious implications for data quality.ConclusionsThe image processing issues identified will often pass unnoticed by an analysis of the standard data returned from an experiment. We detail some simple diagnostics that can be implemented to identify problems of this nature, and outline approaches to correcting for such problems. These approaches require access to the raw data from the arrays, not just the summarized data usually returned, making the acquisition of such raw data highly desirable.

MicroRNA/Argonaute 2 regulates nonsense‐mediated messenger RNA decay

Imperfect base-pairing between microRNA (miRNA) and the 3'-untranslated region of target messenger RNA (mRNA) triggers translational repression of the target mRNA. Here, we provide evidence that human Argonaute 2 targets cap-binding protein (CBP)80/20-bound mRNAs and exon junction complex-bound mRNAs and inhibits nonsense-mediated mRNA decay (NMD), which is restricted tightly to CBP80/20-bound mRNAs. Furthermore, microarray analyses reveal that a subset of cellular transcripts, which are expected to be targeted for NMD, is stabilized by miRNA-mediated gene silencing. The regulation of NMD by miRNAs will shed light on a new post-transcriptional regulation mechanism of gene expression in mammalian cells.

Abstract 1714: Transcriptional networks downstream of the AR identify clinically relevant prostate cancer targets

Abstract The AR signalling axis is central to Prostate Cancer (PrCa) biology, shown by its use in both screening (PSA) and treatment of the disease (androgen deprivation therapy). Identifying direct target genes of the AR will provide a better understanding the key signalling pathways controlled by the AR in PrCa and could lead to improved biomarkers and future therapeutic targets for the disease. To this end we have performed fine mapping of androgen responsive gene expression in prostate cancer cell lines using Illumina bead-arrays, coupled with AR chromatin-immunoprecipitation and Solexa sequencing technology (ChIP-seq). This approach generated over ten thousand candidate AR target genes. We have then undertaken large scale, cross-platform validation using BioTrove Realtime PCR panels which allow the measurement of &amp;gt;600 transcripts simultaneously. Using this combination of approaches we have identified and validated several hundred AR regulated genes. We found that metabolic enzymes and kinases were significantly enriched in the set of direct AR regulated genes. In nine independent clinical gene expression studies we found that the most consistently up-regulated AR target gene was a calcium regulated kinase. At the protein level this kinase showed increased expression in two independent patient cohorts. A small molecule inhibitor of this kinase reduced the growth of a panel of PrCa cell lines in vitro and tumour growth in xenograft models. Interestingly, we found that the levels of this AR regulated kinase were reduced in clinical PrCa following androgen deprivation therapy, but were raised in Castrate Resistant disease. Therefore, we have identified an AR regulate kinase which contributes to tumour growth and is a potential marker and therapeutic target in both hormone naïve and anti-androgen resistant disease. In summary, these data show that combining expression analysis, ChIP-seq and high through-put validation has identified a framework to understand the oncogenic functions of the AR in prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1714.