Expression data from SPAK transfected Caco2-BBE cells
ABSTRACT: Ste20-related proline/alanine rich kinase (SPAK) is involved in diverse function, such as the pathogenesis of inflammatory bowel diseases. It is very important to study the underlying mechanisms by which SPAK regulates the progress of IBD. We used microarrays to detail the global programme of gene expression underlying cellularisation and identified distinct classes of up-regulated genes during this process. We transfected colon cancer modified cell line Caco2-BBE cells with SPAK plasmid and vector for RNA extraction and hybridization on VMSR Human 30k oligonucleotide microarrays.
Project description:To determine the effect of prohibitin overexpression on global gene expression in Caco2-BBE intestinal epithelial cells. 4 individual wells of Caco2-BBE cells, passage 41, were transfected with either empty vector (pcDNA4) or prohibitin/pcDNA4 for 72 hours. Total RNA isolated from 4 wells of cells/per treatment were pooled together for labeling and hybridization purposes.
Project description:In this study, we examined Caco-2 cell gene expression after infection with E. coli (Ec), Lactobacillus plantarum (Lp) and the combination of the two (mix) Cells were washed in PBS and re-fed with experimental DMEM without serum or antibiotics before the experiments. Caco-2 cells were infected with E. coli (10 9 CFU/ml at 1:10 multiplicity ratio), and/or L. plantarum (1010 CFU/ml, 1:100 multiplicity ratio), and incubated for 2 hr
Project description:Zinc (Zn) is an essential trace element for all life forms. Zn supplementation has been used to treat diarrheal disease in children, and in the U.S. swine industry at pharmacological levels to promote growth and fecal consistency, but underlying mechanisms explaining these beneficial effects remain unknown. Thus, we hypothesized that the benefits of pharmacological Zn supplementation were a result of changes in gene expression. For this study, liver RNA from newly weaned pigs fed dietary Zn as Zn oxide for 14 days at either adequate (150 Zn/kg) or pharmacological (2000 mg Zn/kg) levels was evaluated using a 70-mer oligonucleotide microarray. Interrogation of this microarray revealed 658 annotated transcripts (FDR ≤ 0.05) affected by pharmacological Zn supplementation. Relative real-time RT-PCR was used to confirm differential expression of two genes. Results suggest that feeding pharmacological Zn (2000 mg Zn/kg) affects genes involved in reducing oxidative stress and in amino acid metabolism, which are essential for cell detoxification and proper cell function. Oligonucleotide microarrays used for this study consisted of 13,297 70-mer oligos (Pig Array-Ready Oligo Set v. 1.0 and Pig Oligo Extension Set v. 1.0, Qiagen, Inc., Valencia, CA) each spotted once on a single slide. Slides were printed at the Michigan State University Research Technology Support Facility. Oligonucleotides spotted in multiple locations for use as potential controls included 76 Arabidopsis thaliana gene spots, 17 beta tubulin spots, 17 glyceraldehyde-3-phosphate dehydrogenase spots, 85 heat shock protein gene spots, 69 ribosomal protein gene spots, 112 randomly generated negative control spots and 470 blanks. The microarray was screened with the Zn150 and Zn2000 samples, and four microarray slides were screened. Zn150 samples were randomly paired with Zn2000 samples. Two samples from each treatment were labeled with Cy3 and the other two were labeled with Cy5.
Project description:Transcripts upregulated or downregulated by overexpression of N-terminal G3BP were identified through expression profiling of a total of 12,135 genes in comparison with S2-013 clones in which N-terminal G3BP is overexpressed and control clones. We endeavored to identify factors and pathways regulated by CD24 and to obtain leads regarding the mechanism underpinning the observed phenotypic changes by performing cDNA microarray analysis on two S2-013 CD24 RNAi clones and two control clones. Total RNAs from two clones of S2-013 CD24 RNAi or control cells were mixed and used for cDNA microarray analysis.
Project description:We used microarray to determine the miRNA whose expression was changed at 6 hours after inflammatory cytokines (TNFα, IL1α, IL1β, or IL6) treatment. Two-condition experiment; Caco2 control vs Caco2 treated with TNFα, IL1α, IL1β,or IL6 for 6 hours
Project description:Analysis of the genome-wide transcrptional effects caused by the deletion of the IWR1 gene by comparing the transcriptional profile of a iwr1 mutant strain with the isogenic wild type strain when cells were exponentially grown in YPD medium. Three independent cultures for the wt and the iwr1 mutant were used for the whole genome transcription analysis and they were grown in YPD at the early exponential phase. Total RNA was isolated and cDNA synthesis and labeling, filter hybridization and quantification/normalization of hybridization signals were performed as described in Garcia-Martinez, J., Aranda, A. and Perez-Ortin, J.E. (2004) Mol Cell 15(2),303-313.
Project description:Sessile serrated adenomas are now recognized as precursor lesions of a substantial subset of colorectal cancers arising via a so-called “serrated pathway”. However, their biological markers remain to be defined. The aim of our study was to identify differentially expressed genes in sessile serrated adenomas, hyperplastic polyps and tubular adenomas. Gene expression analysis demonstrated molecular differences between polyp types. Further studies using QRT-PCR on Cathepsin E demonstrated a significantly (p< 0.05) higher expression in sessile serrated adenomas as compared to both other polyp types. Trefoil Factor 1, showed the same trend of expression for sessile serrated adenomas as compared to hyperplastic polyps, and was significantly higher in both polyps compared to tubular adenomas. Immunohistochemistry for both proteins demonstrated strong cytoplasmic staining of abnormal crypts in all sessile serrated adenomas while staining in tubular adenomas and hyperplastic polyps was weak and focal. BRAF and KRAS mutation analysis were employed to further validate polyp discrimination. The findings demonstrated the positive association of the BRAF mutation, V600E, with sessile serrated adenomas and KRAS mutations with tubular adenomas (P<0.05). This study demonstrates CTSE and TFF1 over-expression in sessile serrated adenomas compared to both hyperplastic polyps and tubular adenomas. Keywords: colonic polyp tissue comparison, linear modelling, SSA Microarray analysis was performed on 13 SSAs and 11 TAs. SSAs were from 4 males and 9 females (mean age of 75) and TAs were from 5 males and 6 females (mean age of 72). Samples were directly hybridised to each other (SSA versus TA) or to a pooled normal control (SSA versus control). A linear model (Smyth 2004) was fitted to the data bringing together the three contrasts: SSA versus control, TA versus control and SSA versus TA. Human OligoLibrary (Compugen Human Oligo Library (v1) containing 18861 60-mer oligonucleotides, representing approximately 16,000 unique genes) by the Adelaide Microarray Centre (Australia) was used. Slides were scanned using a GenePix 3000B scanner (Axon Instruments, Sunnydale, CA), and the Spot package (CSIRO, Australia) was used to identify spots and estimate fore- and background intensities (using a morphological opening background estimator) (Yang, Buckley et al. 2001; Ritchie 2004). Data analysis was performed in R (www.r-project.org) using the Limma package of Bioconductor (Gentleman, Carey et al. 2004; Smyth 2004). Loess print tip method was used to correct for dye-bias and intensity within each group of adjacent spots printed by one pin (Yang, Dudoit et al. 2002). Linear modelling was performed with the Limma package of Bioconductor (Smyth 2004).
Project description:To study the mechanisms of Ni resistance in the metal resistant Acidiphilium sp. PM, the transcriptome of Acidiphilium sp. PM was studied 5min and 30 min after the addition of 10mM Ni and compared to the transcriptome in untreated cells. Two-condition hybridization experiments: untreated cells vs cells treated with 10mM Ni (for either 5 or 30 minutes). Eighteen 100ml-cultures: 6 untreated control cultures (not exposed to Ni), 6 cultures exposed to 10mM Ni for 5min, and 6 cultures exposed to 10mM Ni for 30 min. Cultures were independently grown and harvested. Gene expression levels of one untreated replicate were compared separately with one 5min-exposed replicate and with one 30min-exposed replicate (yielding two microarray data sets. One for each hybridization).
Project description:We have constructed a rainbow trout high-density oligonucleotide microarray by using all the available tentative consensus (TC) sequences from the Rainbow Trout Gene Index database (The Computational Biology and Functional Genomics Lab., Dana Farber Cancer Institute and Harvard School of Public Health). The Rainbow Trout Gene Index integrates research data from all available international rainbow trout genomic research projects. The newly designed microarray incorporates 37,394 unique transcript-specific oligonucleotide probes, 60-mer long each. The microarray was printed according to our design by Agilent Technologies using the 4 X 44-design format and contains 1417 Agilent control spots. The performance of the new microarray platform was evaluated by analyzing gene expression associated with the rainbow trout vitellogenesis-induced muscle atrophy. These chips can be ordered from Agilent using design number 016320. This microarray is anticipated to open new avenues of research that will aid in the development of novel strategies to enhance growth efficiency and quality in salmonid species. Keywords: Development of an oligo-array for rainbow trout The performance of the new microarray platform was evaluated by analyzing transcriptome response associated with the rainbow trout vitellogenesis-induced muscle atrophy. Severe muscle deterioration accompanies the physiological responses of the energetic demands of the rainbow trout spawning/vitellogenesis. Atrophying muscle of fertile fish had 11% less extractable muscle (g/bw) and 11% less protein content compared to non-atrophying muscle of sterile fish (p<0.01). The rainbow trout was used to profile changes in gene expression of atrophying muscles. Gene expression levels were determined by comparing the amount of mRNA transcript present in the experimental sample (fertile fish) to the control (sterile fish). RNAs isolated from each experimental fish were run on separate microarrays in independent experiments, with no pooling. A total of 8 fish were used in the microarray experiments (4 replicates x 2 groups). Fluorophors (Cy3 and Cy5) were randomly assigned to RNA from each of the atrophying and nonatrophying muscles to limit the dye effect.
Project description:Stripe rust, caused by Puccinia striiformis f. sp. tritici, is a destructive disease of wheat worldwide. Genetic resistance is the preferred method for controlling stripe rust, of which two major types are race-specific and race non-specific resistance. Race-specific resistance includes the qualitatively inherited all-stage resistance, controlled by single major resistance (R) genes. Conversely, adult-plant resistance is race non-specific, inherited quantitatively, and is durable. Previously, we characterized the gene expression signatures involved in Yr5-controlled all-stage resistance and Yr39-controlled adult-plant resistance using the Affymetrix Wheat GeneChip. For this study, we designed and constructed custom oligonucleotide microarrays containing probes for the 116 and 207 transcripts that we had found important for the Yr5 and Yr39 resistance responses, respectively. We used this custom microarray to profile the resistance responses of eight wheat genotypes with all-stage resistance (Yr1, Yr5, Yr7, Yr8, Yr9, Yr10, Yr15, and Yr17). The aim of this analysis was to identify common and unique gene expression signatures involved in race-specific resistance accross genotypes, which were used to infer information regarding the general pathways involved in all-stage resistance. Keywords: Stress response Eight wheat genotypes with defined single gene all-stage resistance to particular stripe rust (Pst) isolates were selected for this study, as well as the Pst susceptible genotype ‘Avocet Susceptible’ (AVS). Near Isogenic Lines (NILs) for each of eight Yr resistance genes (Yr1, Yr5, Yr7, Yr8, Yr9, Yr10, Yr15 and Yr17) were developed at the Plant Breeding Institute, Sydney, Australia, by backcrossing the Yr gene donors with the recurrent susceptible spring wheat genotype (Triticum aestivum L.) AVS six times (AVS*6/Yr*) and selecting for the appropriate resistance at each generation. Both virulent and avirulent isolates of Pst were selected for each NIL, except for Yr15, for which no virulent isolate is currently known. Care was taken to select the fewest isolates needed to satisfy the spectrum of virulence/avirulence required, resulting in the use of six isolates identified as PST-17, PST-21, PST-43, PST-45, PST-78 and PST-AUS. Each isolate was selected and maintained on susceptible genotypes. For each of three biological replications, individual genotypes were planted in separate 25 X 42.5-cm flats using a potting mix (6 peat moss: 4 vermiculite with lime: 3 sand: 3 commercial potting mix: 2 perlite: 1.7 g/L lime: 3.3 g/L Osmocote: 2.2 g/L ammonium nitrate). Each flat consisted of three rows of six seedlings, with rows randomly assigned one of two harvest times (24 and 48 h post-inoculation). Seedlings from the 3rd row were used to monitor the expected disease responses to inoculation. Seedlings were grown to the second leaf stage (Feekes 1.2, emergence with second leaf unfolded, ~10 days after planting) in a greenhouse with a diurnal temperature cycle of 10ºC (2:00 am) to 25ºC (2:00 pm) and a 16 h light/8 h dark cycle. Inoculation was performed by misting the plants with sterile water and applying a 1:20 urediniospore:talc mixture to leaves with a sterile brush. Talc was used to aid in the spread and adhesion of spores over leaf surfaces. Control flats were treated the same way except for the absence of spores in the talc. All treatments for each biological replication were performed at 9 am Pacific Standard Time. To promote spore germination, all flats were transferred to a dew chamber (100% RH) operating at 10ºC in the dark for 24 h, before being placed in a growth chamber with a diurnal temperature cycle of 4ºC (2:00 am) to 20ºC (2:00 pm) and a 16 h light/8 h dark cycle. Rows of plants were harvested from all flats at the assigned times for RNA extraction.