SSHscreen analysis of cowpea drought stress SSH libraries (F&R) to calculate Enrichment ratio 2 and 3 values
ABSTRACT: Screening cDNA clones from SSH library by hybridization with cDNA used to construct the library. Treated samples were drought-stressed cowpea plants, and control samples were cowpea plants subjected to a standard watering regime. cDNA clones from forward and reverse libraries are spotted on the same array, but data from each library were analysed separately after normalization using SSHscreen software (http://microarray.up.ac.za/SSHscreen ) to calculate Enrichment Ratio 3 (ER3) and Enrichment Ratio 2 (ER2) values for each clone. ER3 is a measure of differential expression, and was determined using a set of hybridizations with unsubtracted treated (UT) and unsubtracted control (UC) cDNA. ER2 is a measure of the relative abundance of a clone's transcript in the original tester sample, relative to other transcripts in the sample prior to the SSH process. ER2 for the forward library was determined using a set of hybridizations with subtracted treated (ST) and unsubtracted treated (UT) cDNA. ER2 for the reverse library is determined using a set of hybridizations with subtracted control (SC) and unsubtracted control (UC) cDNA. Direct comparison of UT and UC cDNAs to calculate ER3 values for F and R library, including dye swaps and replicate arrays. Direct comparison of ST and UT cDNAs to calculate ER2 values for F library, including dye swaps and replicate arrays. Direct comparison of SC and UC cDNAs to calculate ER2 values for R library, including dye swaps and replicate arrays.
Project description:Screening cDNA clones from SSH library by hybridization with cDNA used to construct the library. Treated samples were drought-stressed cowpea plants, and control samples were cowpea plants subjected to a standard watering regime. cDNA clones from forward and reverse libraries are spotted on the same array, but data from each library were analysed separately after normalization using SSHscreen software (http://microarray.up.ac.za/SSHscreen ) to calculate Enrichment Ratio 3 (ER3) and Enrichment Ratio 2 (ER2) values for each clone. ER3 is a measure of differential expression, and was determined using a set of hybridizations with unsubtracted treated (UT) and unsubtracted control (UC) cDNA. ER2 is a measure of the relative abundance of a clone's transcript in the original tester sample, relative to other transcripts in the sample prior to the SSH process. ER2 for the forward library was determined using a set of hybridizations with subtracted treated (ST) and unsubtracted treated (UT) cDNA. ER2 for the reverse library is determined using a set of hybridizations with subtracted control (SC) and unsubtracted control (UC) cDNA. Overall design: Direct comparison of UT and UC cDNAs to calculate ER3 values for F and R library, including dye swaps and replicate arrays. Direct comparison of ST and UT cDNAs to calculate ER2 values for F library, including dye swaps and replicate arrays. Direct comparison of SC and UC cDNAs to calculate ER2 values for R library, including dye swaps and replicate arrays.
Project description:Background & Aims: Genome-wide gene expression (GWGE) profiles of mucosal colonic biopsies have suggested the existence of a continuous inflammatory state in quiescent ulcerative colitis (UC). The aim of this study was to use DNA microarray-based GWGE profiling of mucosal colonic biopsies and isolated colonocytes from UC patients and controls in order to identify the cell types responsible for the continuous inflammatory state. Methods: Adjacent mucosal colonic biopsies were obtained endoscopically from the descending colon in patients with active UC (n=8), quiescent UC (n=9), and with irritable bowel syndrome (controls, n=10). After isolation of colonocytes and subsequent extraction of total RNA, GWGE data were acquired using Human Genome U133 Plus 2.0 GeneChip Array (Affymetrix, Santa Clara, CA). Data analysis was carried out by principal component analysis and projection to latent structure-discriminant analysis using the SIMCA-P11 software (Umetrics, Umeå, Sweden). Results: A clear separation between active UC, quiescent UC and control biopsies were found, whereas the model for the colonocytes was unable to distinguish between quiescent UC and controls. The differentiation between quiescent UC and control biopsies was governed by unique profiles containing gene expressions with significant fold changes. These primarily belonged to the family of homeostatic chemokines revealing a plausible explanation to the abnormal regulated innate immune response seen in patients with UC. Conclusion: This study has demonstrated the presence of a continuous inflammatory state in quiescent UC, which seems to reflect an altered gene expression profile of lamina propria cells. Keywords: Colonocytes, continuous inflammation, mucosal colonic biopsies, gene expression profiles Adjacent mucosal colonic biopsies were attained endoscopically from the descending colon in patients with active UC (n=8), quiescent UC (n=9), and in controls (n=10). After extraction of total RNA, genome-wide gene expression data were acquired using Human Genome U133 Plus 2.0 GeneChip Array (Affymetrix, Santa Clara, CA). Amplification was required to obtain sufficient amounts of labelled complementary RNA (cRNA) target for analysis with arrays. Data analysis was carried out by principal component analysis and projection to latent structure-discriminant analysis using the SIMCA-P11 software (Umetrics, Umeå, Sweden).
Project description:Spec-seq directly mesaures specificity by sequencing. We run an EMSA experiment on TF of interest with a library of DNA targets. Then we extract and sequence the bound and unbound portion of the DNA separately. Finally we count the occurences of each varient in the library and calculate the ratio and the relative binding energy. Overall design: 15 independent Spec-seq experiments
Project description:Background and aims: Mucosal abnormalities are potentially important in the primary pathogenesis of ulcerative colitis (UC). We investigated the mucosal transcriptomic expression profiles of biopsies from patients with UC and healthy controls (HC), taken from macroscopically non-inflamed tissue from the terminal ileum and three colonic locations with the objective of identifying abnormal molecules that might be involved in disease development. Methods: Whole-genome transcriptional analysis was performed on intestinal biopsies taken from 24 UC, 26 HC and 14 patients with Crohn’s disease. Differential gene expression analysis was performed at each tissue location separately and results were then meta-analysed using Fisher’s method. Significantly differentially expressed genes were validated using qPCR. Gene location within the colon was determined using immunohistochemistry, subcellular fractionation, electron and confocal microscopy. DNA methylation was quantified by pyrosequencing. Results: Seven probes were abnormally expressed throughout the colon in UC patients with Family with sequence similarity member 5 C (FAM5C) being the most significantly underexpressed. Attenuated expression of FAM5C in UC was independent of inflammation, unrelated to phenotype or treatment, and remained low at rebiopsy approximately 23 months later. FAM5C is localised to the brush border of the colonic epithelium and expression is influenced by DNA methylation within its promoter. Conclusion: Genome-wide expression analysis of non-inflamed mucosal biopsies from UC patients identified FAM5C as significantly under-expressed throughout the colon in a major sub-set of patients with UC. Low levels of this gene could predispose to or contribute to the maintenance of the characteristic mucosal inflammation seen in this condition. Total RNA was extracted from the intestinal biopsies taken from macroscopically normal mucosa in the rectum, descending colon, ascending colon and terminal ileum in clinically quiescent Ulcerative colitis and Crohn's disease patients and compared to healthy controls. Normalized data for 26,261 probes out of 47,323 only. Criteria for inclusion not specified. The non-normalized matrix contains the complete non-normalized data for all probes.
Project description:UC pouchitis is a potential model of UC. We prospectively examined the pouch transcriptomes of UC and familial adenomatous polyposis (FAP) IPAA patients to unveil molecular mechanisms of UC pouchitis susceptibility. Methods: Total RNA was isolated using the AllPrep DNA/RNA Mini Kit (QIAGEN, Cat No. 8020). RNA quality was evaluated using Bioanalyzer (Agilent, Santa Clara, CA). All RNA samples displayed RNA Integrity Number (RIN) >7. RNAseq including cDNA library preparation was processed at the Genomics Core Facility of University of Chicago (https://fgf.uchicago.edu/). Total RNA in the amount of 100-500μg per sample was depleted of ribosomal RNA using the Ribo-Zero kit (Epicentre, Madison, WI). The directional (first strand) cDNA libraries were prepared following the guide of TruSeq Stranded Total RNA Sample Preparation kit. Results: Unlike FAP patients, UC subjects exhibited a large set of differentially expressed genes (DEGs) between pouch and pre-pouch mucosa as early as 4 months after pouch functionalization. Functional pathway analysis of DEGs in UC pouch revealed: (1) Gain of colon-associated gene expressions and loss of ileum associated gene expressions, (2) enhanced state of immune/inflammatory response, and (3) suppressed xenobiotic, lipid, and bile acid metabolic pathways. These changes were corroborated upon reanalysis of a published larger cross-sectional study of UC and FAP patients. Moreover, >70% of DEGs mapped to published IBD and normal colonic microarray datasets displayed directional changes consistent with active UC, but not Crohn's disease. Conclusions: UC patients exhibit a unique transcriptomic response to ileal pouch creation that can be observed well before disease. The transcriptome alterations provide insights into pouchitis Overall design: Seventeen patients with UC and four patients with FAP were recruited at the University of Chicago and the Mayo Clinic Rochester. All patients underwent a total proctocolectomy with ileal pouch anal anastomosis (IPAA) as a standard of care. UC patients underwent a pouchoscopy for biopsy of the pre-pouch ileum and pouch at 4 months, 8 months, and 12 months after ileostomy closure. None of these patients had pouchitis.
Project description:Chromosomal instability is a hallmark of cancer and genes that display abnormal expression in chromosomally aberrant regions are likely to be key players in tumor progression. Identifying such driver genes from high-throughput data requires computational methods that are capable of integrating data from several sources and thereby enhance the reliability of driver gene identification. Hence, several algorithms that integrate copy number and expression data have been developed but their relative performance has not been assessed so far. We have compared 10 algorithms that integrate high-throughput copy number and transcriptomics data using simulated, cancer cell line and primary tumor data. Our results show that there are significant differences between the methods and their performance decreases significantly with small sample sets. Head and neck squamous cell carcinoma (HNSCC) cell lines from the tongue (UT-SCC-21,UT-SCC-24B, UT-SCC-30, UT-SCC-67, UT-SCC-73, UT-SCC-76A, UT-SCC-81, UT-SCC-87,UT-SCC-95) and larynx (UT-SCC-8, UT-SCC-11, UT-SCC-75) were provided by the Department of Otorhinolaryngology-Head and Neck Surgery at the Turku University Central Hospital (Turku, Finland). HNSCC cell lines SCC-4, SCC-9, SCC-25 and human skin keratinocyte HPV-16 E6/E7 transformed cell line CCD1106 KERTr was ordered from American Type Culture Collection (ATCC; Manassas, VA) and cultured according to the ATCC recommendations.
Project description:The bacterial pathogen Erwinia amylovora is the causal agent of fire blight, an economically significant disease of apple and pear. Disease initiation by E. amylovora requires the translocation of effector proteins into host cells by the hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS). The alternate sigma factor HrpL positively regulates the transcription of structural and translocated components of the T3SS via hrp promoter elements. To characterize genome-wide hrpL-dependent gene expression in E. amylovora Ea1189, wild-type and Ea1189hrpL strains were cultured in hrp-inducing minimal medium, and total RNA was compared using a custom microarray designed to represent the annotated genes of E. amylovora ATCC 49946. Results revealed 24 genes differentially-regulated in Ea1189hrpL compared to Ea1189 with fold-change expression ratios greater than 1.5; of these, the expression of 19 genes was up-regulated while five genes exhibited negative regulation. To expand our understanding of the HrpL regulon and to elucidate direct versus indirect hrpL-mediated effects on gene expression, the genome of E. amylovora ATCC 49946 was examined in silico using a hidden Markov model assembled from known Erwinia spp. hrp promoters. This technique identified 21 putative type III novel hrp promoters; of these, 8+ were validated with quantitative polymerase chain reaction based on expression analyses. In total, hrpL-regulated genes encode all known components of the hrp T3SS and 5 putative type III effectors. Eight genes displayed apparent indirect hrpL regulation suggesting that the hrpL regulon is connected to downstream signaling networks. Construction of deletion mutants of three novel hrpL-regulated genes has resulted in the identification of additional virulence factors in E. amylovora as well as mutants displaying abnormal motility and biofilm phenotypes.
Project description:The Del-Mar 14K chip was used to interrogate differential expression of transcripts in the white isthmus (WI) compared with the adjacent magnum (Mg) and uterine (Ut) segments of the hen oviduct. Differential expression of genes common to both comparisons (WI/Mg and WI/Ut) was detected for 204 annotated proteins. Of these, 58 genes were overexpressed in both WI/Mg and WI/Ut, and are therefore considered to be the most interesting candidates for WI - specific functions. Additionally, general analysis revealed 135 clones hybridizing to overexpressed transcripts (WI/Mg + WI/Ut), and corresponding to 102 NCBI annotatated non-redundant Gallus gallus gene ID~s. This combined analysis revealed that structural proteins highly over-expressed in white isthmus were collagen X (COL10A1), Fibrillin (FBN1) and Cysteine Rich Eggshell Membrane Protein (CREMP). In addition, genes encoding collagen-processing enzymes were over-expressed, as were proteins known to regulate disulfide cross-linking, suggesting that coordinated upregulation of gene networks in the white isthmus is associated with eggshell membrane fibre formation. IPA interactome analysis reinforces the key role of the estrogen receptor and SMAD3 in mediating gene regulation during eggshell membrane synthesis. These results will assist with development of selection strategies to improve eggshell quality and food safety of the table egg. Keywords: Laying hen, eggshell, oviduct, Isthmus expression, cDNA microarray, indirect cDNA labelling, Alexa Fluor dyes Keywords: Expression profiling by array A balanced block hybridization design (Dye switch) was used where half of the samples were labelled with Alexa® 555 fluorescent dye and the other half with Alexa® 647. A total of 16 microarray slides were used for hybridization to 32 samples that correspond to four tissue contrast (White isthmus versus magnum and uterus versus white isthmus).
Project description:CUTO-3 and KM12-luc cell lines were treated with DMSO or 1 uM CH7057288 for 4 and 24 hours. Cellular RNA was extracted, processed to generate an mRNA library, and sequenced from both ends of the library using HiSeq 2500 Sequencing System. RSEM software was used to align reads against RefSeq transcripts and calculate expression values (FPKM) for each gene. Overall design: mRNA expression analysis of two cell lines treated with TRK inhibitor was performed by RANA-seq Raw data has not been submitted due to patient privacy laws in Japan.
Project description:delta-Np63 is highly expressed in squamous cell carcinoma of the head and neck (HNSCC). To evaluate its function in HNSCC we depleted delta-Np63 by siRNAs in the HNSCC cell line UT-SCC-74A. The transcriptome was analysed by cDNA microarray.