Project description:The aim was to examine changes in gene expression of the endometrium exposed to long-term tamoxifen treatment in comparison to age matched controls. To achieve this, endometrial tissues were obtained from women receiving tamoxifen treatment who were undergoing a hysterectomy. Using cDNA microarrays, gene expression changes in the postmenopausal endometrium of these women was compared with that in endometrium of age matched women not receiving tamoxifen. Endometrial tissue from post-menopausal women was obtained following ethical approval from the Leicester NHS Trust. None of the women had received any hormonal treatment for two months prior to the procurement of the specimens. Tissues were taken from untreated women (n=6) or those treated for 4 to 5 years with tamoxifen (20mg/day) (n=4), aged 58-82 (65 ± 9.1, mean ± SD). Total RNA was extracted. Controls were pooled. RNA labelling, hybridisation and analysis of fluorescence was carried out as described by Turton et al (2001). Cy3/Cy5 Dye swap labelling was carried out on samples from each patient. Reference: Turton NJ et. al. (Oncogene (2001) 20, 1300-1306
Project description:To identify substrates of the ubiquitinating E3 enzyme Rsp5 we applied purified Rsp5 to duplicate protein arrays. The Rsp proteins were expressed as fusion proteins to GST. We used as a control Ubr1, a RING domain containing E3 ligase We analyzed Rsp5 from S.cerevisiae on duplicate arrays, with four control chips, two without Rsp5 and two with Ubr1.
Project description:Circulating blood proteomics enables minimally invasive biomarker discovery. Nanoparticle-based circulating plasma proteomics studies have reported varying number of proteins (ca 2000-7000), but it’s unclear whether higher protein number is more informative. Here, we first develop OmniProt – a silica-nanoparticle workflow optimized through systematic evaluation of nanoparticle types and protein corona formation parameters. Next, we present an Astral spectral library for 10,109 protein groups. Using Astral with 60 sample-per-day throughput, OmniProt identifies ca 3000 to 6000 protein groups from human plasma. Notably, platelet/erythrocyte/coagulation-related contamination artificially elevates protein identifications and compromises quantification accuracy in nanoparticle-enriched samples. Through controlled contamination experiments, we identified biomarkers for platelet/erythrocyte/coagulation-related contaminations in nanoparticle-based plasma proteomics. We developed open-access software Baize for contamination assessment. We validated pipeline in 193 patients with CT-indistinct benign nodules or early lung cancers, flagging five contaminated samples. This study reveals contamination alters protein identification/quantification in nanoparticle-based plasma proteomics and presents Baize software to evaluate contamination.
Project description:The current study reports a genome-wide transcriptional profiling of the only known GM-NM-1 mutants in rice (RGA1). Total RNA was isolated from the leaves of 25 day old rice d1 (GM-NM-1) mutant (HO-541) and its corresponding wild type (Nipponbare) grown on B-5 agar media.Total RNAs from the rice GM-NM-1 mutant and wild type were first converted into cDNAs, from which labeled cRNAs were generated with Cy3 and Cy5 dyes and hybridized with 44K 60-mer whole genome rice microarrays on glass slides.The slides were washed and scanned on an Agilent scanner (G2565B) at 100 % laser power and data extracted with Agilent Feature Extraction software (version 9.1). The experiment was repeated with a biological-cum-technical replicate, where total RNA samples from a fresh batch of rice (mutant and WT) plants were labeled with opposite dyes (dye-swap) and used for microarray analysis.
Project description:The aim of this study was to construct a prediction model for axillary lymph node metastasis (ALNM) using a DNA microarray assay for gene expression in breast tumor tissues. Luminal A breast cancers, diagnosed by PAM50 testing, were analyzed, and a prediction model (genomic nodal index (GNI)) consisting of 292 probe sets for ALNM was constructed in a training set of patients (n=388), and was validated in the first (n=59) and the second (n=103) validation sets. AUCs of ROC were 0.820, 0.717, and 0.749 in the training, first, and second validation sets, respectively. GNI was most significantly associated with ALNM, independently of the other conventional clinicopathological parameters in all cohorts. It is suggested that GNI can be used to identify the patients with a low risk for ALNM so that sentinel lymph node biopsy can be spared safely. This DATA set: J03 contains 120 (n+ 60, n- 60) samples of the above first and second validation sets from Japan (OUH_1,2). A long time passed, and now it is unclear how these 120 cases were distributed among the first and second validation sets. *Note: This old data has been updated multiple times by others. Then, there are some differences from the original 2014 paper and unclear points still remain. Therefore, do not use it for formal analysis aimed at public insurance coverage etc. This is for research purposes only. Please cite this paper when writing a new paper. PMID: 25016059 DOI: 10.1016/j.canlet.2014.07.003
Project description:This SuperSeries is composed of the following subset Series: GSE24037: Salivary cytokine alterations in HIV infection part 1 GSE24064: Salivary cytokine alterations in HIV infection part 2 Refer to individual Series
Project description:We have designed a zebrafish genomic microarray to identify DNA-protein interactions in the proximal promoter regions of over 11,000 zebrafish genes. Using these microarrays, together with chromatin immunoprecipitation with an antibody directed against tri-methylated lysine 4 of Histone H3, we demonstrate the feasibility of this method in zebrafish. This approach will allow investigators to determine the genomic binding locations of DNA interacting proteins during development and expedite the assembly of the genetic networks that regulate embryogenesis. Genomic array design Microarrays were designed as described below and manufactured by Agilent Technologies (www.agilent.com). Further information on design can be found at http://jura.wi.mit.edu/bioc/gbell/zfish_chip/. Selection of transcription start sites and identification of promoter sequences We interrogated 5 databases: Ensembl, VEGA, Refseq, ZGC full length clones and a database provided by Dr. Leonard Zon (Harvard Medical School, Boston, USA) in order to assemble an extensive list of zebrafish transcripts. The Zon lab database is a hand-curated database of zebrafish genes that have homologues in other species. We included all transcripts that appeared in the manually annotated databases (VEGA, Zon) and in the ZGC full length database. We also identified genes present in any 2 of the 5 databases and included those not already selected. The transcripts were mapped to the zebrafish genome (Zv4, June 2004) obtained from UCSC Bioinformatics (http://genome.ucsc.edu) and the transcription start site (TSS) for each transcript was determined. Transcripts with TSSs within 500bp were clustered into a transcriptional unit (TU) and promoter regions were identified relative to the most upstream TSS. This resulted in the identification of 13,413 TUs and corresponding promoter regions. Each promoter region was extracted and masked for repetitive sequence by RepeatMasker. If the promoter region contained a gap the upstream sequence was also masked. Information on the transcriptional units that were included in the final design can be found at http://jura.wi.mit.edu/bioc/gbell/zfish_chip/. Selection of oligonucleotides 60-mer oligonucleotide probes representing the region between 1.5kb upstream and 0.5kb downstream of the annotated TSS of each transcriptional unit were then designed. Although transcription factors and other DNA binding proteins are known to regulate genes from distances of greater than -1.5kb or + 0.5kb, much information can be gained from regions close to the TSS [45], and the H3K4Me3 mark studied in this paper is found at the most 5â end of a gene, close to the TSS. Selection of 60-mers for the microarrays was essentially as described in [14] using the Zv4 build of the zebrafish genome and a locally customized version of ArrayOligoSelector. 60-mers were chosen so that promoter regions contained approximately one probe every 250bp with a maximum distance between probes for each promoter region set at 600bp. In cases where only one probe could be designed for a particular TU these were not included in the final design. This process yielded 80,839 probes for 11,171 promoter regions We also incorporated several sets of control probes, both positive and negative. On each array there are 1090 probes designed against âgene desertâ regions, which are genomic regions that are unlikely to be bound by transcriptional regulators, and 270 probes designed against Arabidopsis thaliana genes, which are not present in the zebrafish genome (by BLAST). In addition, because our main motivation for making these microarrays is to identify mesodermally-regulated genes we included 7 genes expressed in mesoderm during gastrulation as positive controls (wnt11, flh, vent, msgn1, myod, fgf8, pcdh8). Probes designed against these promoters, which flank from 3-4kb around each TSS, are arrayed 2-4 times on each slide. Since these genes are expressed at gastrula stages to varying degrees, they also serve as a positive controls in this study. Finally there are 2256 controls added by Agilent and a variable number of blank spots. These probes were divided between two microarray slides each with 44,290 features. We refer to these two microarray slides as the âproximal promoter setâ. A proximal promoter set based on these designs as well as an expanded set of 9 slides which contain regions from â9kb to + 3kb relative to the TSS, are available by contacting Agilent (www.agilent.com) or by downloading the design files from http://jura.wi.mit.edu/bioc/gbell/zfish_chip/ for self-manufacture.
Project description:Gene expression profiling of human promyelocytic cells in response to infection with Anaplasma phagocytophilum. Total RNA derived from 3DPI Anaplasma phagocytophilum-infected HL-60 cells was labeled with A647 and total RNA derived from 3DPI Mock-infected HL-60 cells was labeled with A546. For each, 5 µg of total RNA was labeled using Genisphere Array900, Alexa Fluor dyes and SuperscriptII. Slide scanned with ScanArray Express and images processed with GenePix Pro version 4.0. Normalized log ratios VALUES determined using R-project statistical environment (http://www.r-project.org) and Bioconductor (http://www.bioconductor.org) through the GenePix AutoProcessor (GPAP, http://darwin.biochem.okstate.edu/gpap) website (P. Ayoubi, unpublished results).
Project description:Less than 30% of children with high-risk (HR) metastatic neuroblastoma (NB) show a long survival (Pearson 2000). In order to identify novel molecular prognostic markers useful to better predict patientsM-bM-^@M-^Y relapse risk estimation, we performed genome- and/or transcriptome-wide analyses of 129 stage 4 HR-NBs. This is the largest study for this NB subtype. Children older than 1 year of age at diagnosis were categorized as M-bM-^@M-^\short-survivorsM-bM-^@M-^] (dead of disease within 5 years from diagnosis) and M-bM-^@M-^\long-survivorsM-bM-^@M-^] (alive with an overall survival time > 5 years). A significant correlation of patient survival with the presence of small number of segmental copy number aberrations (CNA < 3) was observed. Thus, within the group of stage 4 HR-NBs, we identified a specific subgroup of patients (those with highest number of CNA) that have a higher risk of progression/recurrence. The complex genomic pattern is an independent prognostic marker, since MYCN oncogene amplification only affects the predictive value of single CNA (i.e., 1p loss, 17q gain). Integrative analysis of genomic and expression signatures demonstrated that fatal outcome is associated with loss of cell cycle control and with progression of tumor due to deregulation of Rho GTPase signaling and genes related to cell motility. Tumors with MYCN amplification showed a lower chromosome instability compared to MYCN-single copy NBs (P=0.0008), dominated by 17q gain and 1p loss. Moreover, our results suggest that the MYCN amplification mainly drives the disruption of neuronal differentiation and the reduction of cell adhesion process involved in tumor invasion and metastasis. For array-CGH profiling, we analyzed 91 samples of metastatic neuroblastomas. All patients were classified as stage 4 and they were older than 1 year of age at time of diagnosis. Regarding the clinical course, 46 were short-survivors (dead of disease within 60 months from diagnosis. Deaths due to toxicity were censored) and 45 were long-survivors (alive with an overall survival time > 60 months). This submission consists of 22 new samples and 69 other cases previously deposited in GEO under Series accessions GSE14109 and GSE25771.