Gene Expression profiling of M. bovis BCG strain under hypoxia
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ABSTRACT:
ORGANISM(S): Mycobacterium bovis BCG
SUBMITTER: Srivastav BS
PROVIDER: E-GEOD-10336 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Objective of the study is to find out the differentially regulated genes of Mycobacterium BCG strain in extended stationary phase comparison to log phase growth and resuscitation phase. Mycobacterium BCG culture was grown in Sauton medium at 37o C without shaking. Cells at A600 0.6-0.8 were harvested as log phase culture. The cells harvested after 5 months incubation at 37oC without shaking became non culturable and were harvested as extended stationary phase cells. The extended stationary phase cells were treated with resuscitation promoting factor (Rpf) from Micrococcus luteus and harvested after 8 days. Cells of this stage were treated as resuscitation phase cells. Gene expression profiling was carried out using Agilent microarray platform. Keywords: Extended stationary phase, Log phase growth and Resuscitation phase. ⢠Organism: Mycobacterium tuberculosis BCG ⢠Slides: Agilentâs Mycobacterium tuberculosis custom array 4x44k (G2514F) AMADID No: 016013 ⢠Labeling kit: Ambion Message Amp II Bacteria a RNA Amplification Kit Cat# 1790 ⢠Labeling Method: T7 promoter based-linear amplification to generate labeled complementary RNA ⢠Total RNA and cRNA Purification Kit: Qiagenâs RNeasy minikit Cat#74104 ⢠Hybridization Kit: Agilentâs In situ Hybridzation kit 5184-3568 ⢠RNA quality was checked using Bioanalyzer. Hybridization protocol 825ng each of labeled control cRNA was mixed with 825ng of treated labelled cRNA, mixed with appropriate amount of blocking buffer and hybridization buffer (Agilent Technologies). The hybridization mix was applied on to the backings and hybridized to custom designed 60mer Mycobacterium tuberculosis microarray using sure hyb chambers at 65degC for 17 hours. Slides were washed with gene expression wash buffer 1 and 2 (Agilent Technologies) followed by Acetonitrile. Scan protocol Laser detection of Cyanine 3 and Cyanine 5 fluorescence is performed using a confocal scanning instrument containing two tuned lasers, which excite Cyanine dyes at the appropriate wavelengths. Description Slides were scanned at 5 micron resolution using Agilent scanner.Automated feature extraction was done using Agilentâs Feature Extraction Software. Data processing Analysis of feature extracted data was done using Agilentâs GeneSpring GX V 7.3.1 software. Pre-processing of the replicate experiment data was done to flag the outliers. Normalization of the data was done using per spot per chip intensity dependant lowess normalization and dye swap experiment was data transformed. Genes with log2 ratio of 1.29 and above in replicate experiments were considered as up regulated and 0.81 and below was considered down regulated. p-value was calculated by GeneSpring GX for each gene on the basis of replicate probes to indicate statistical significance. Biological significance of differentials were analyzed using Genotypics Biointerpreter - Web based tool for biological interpretation of given genelist.
2010-05-16 | E-GEOD-15642 | biostudies-arrayexpress
Project description:Retinoblastoma Y79 cell line was treated with specific siRNA to silence EpCAM gene expression in vitro and RNA was isolated for microarray experiment to study the changes in the whole gene expression profiling in the Y79 cell line. The study identified 465 up-regulated genes (>1.0 fold) and 205 down-regulated genes (<0.5 fold) in response to knockdown of Ep-CAM. Organism used: Homo Sapiens ï Slides: Whole genome Human 4x44k array ï Starting material: Cells in RNA Later ï RNA Samples used: Control, siRNA1 ï Labeling kit: Agilent Low Input RNA Linear amplification kit ï Labeling Method: T7 promoter based-linear amplification to generate labelled complementary RNA ï Total RNA and cRNA Purification Kit: Qiagenâs RNeasy minikit ï Hybridization Kit: Agilent Insitu Hybridization Kit Hybridization protocol The fragmented cRNA were mixed with 25ul of 2x GE Hybridization Buffer (Agilent). About 45ul of the resulting mixture was applied to the Microarray and hybridized at 65degC for 17 hours in an Agilent Microarray Hybridization Chamber (SureHyb: G2534A) with Hybridization Oven. After hybridization, slides were washed with Agilent Gene expression Wash Buffer I for 1 minute at room temperature followed by a 1 min wash with Agilent Gene expression Wash Buffer II for 37C. Slides were finally rinsed with Acetonitrile for cleaning up and drying. Scan protocol Laser detection of Cyanine 3 and Cyanine 5 fluorescence is performed using a confocal scanning instrument containing two tuned lasers, which excite Cyanine dyes at the appropriate wavelengths. Description Microarrays were scanned on an Agilent scanner (G2565AA) at 100% laser power, 30% PMT.Data extraction was carried out with Agilent Feature Extraction software (version 9.1), and normalization was done using linear per array algorithm according to the manufacturerâs protocol. Data processing Feature extracted data was analyzed using GeneSpring GX v 7.3.1 software from Agilent. Normalization of the data was done in GeneSpring GX using the recommended one color Per Chip and Per Gene Data Transformation: Set measurements less than 0.01 to 0.01, Per Chip: Normalize to 50th percentile, Per Gene: Normalize to Specific Samples. Further quality control of normalized data was done using correlation based condition tree to eliminate bad experiments. Differentially regulated Genes were filtered with cutoff of > 1.5 for Up regulation and < 0.55 for Down Regulation were obtained. Differentially regulated genes were clustered using gene tree to identify significant gene expression patterns.
2010-08-18 | E-GEOD-16991 | biostudies-arrayexpress
Project description:Gene expression analysis study in curcumin treated (20µM curcumin treated Y79 cells ) and control Y79 cells (Suspension Y79 cells). Source were Y79 retinoblastoma cell line from human. Organism used: Homo sapiens ï Slides: Gene Expression Whole Human Genome 4x44k ï Starting material: Cells in RNA later ï RNA Samples used: :Control Y79 and Treated_Curcumin ï Labeling kit: Agilents Low input RNA linear amplification Kit one color Labeling Method: T7 promoter based-linear amplification to generate labeled complementary RNA ï Total RNA and cRNA Purification Kit: Qiagenâs RNeasy minikit ï Hybridization Kit: Agilentâs In situ Hybridzation kit Hybridization protocol The fragmented cRNA were mixed with 25ul of 2x GE Hybridization Buffer (Agilent). About 45ul of the resulting mixture was applied to the Microarray and hybridized at 65degC for 17 hours in an Agilent Microarray Hybridization Chamber (SureHyb: G2534A) with Hybridization Oven. After hybridization, slides were washed with Agilent Gene expression Wash Buffer I for 1 minute at room temperature followed by a 1 min wash with Agilent Gene expression Wash Buffer II for 37C. Slides were finally rinsed with Acetonitrile for cleaning up and drying. Scan protocol Laser detection of Cyanine 3 and Cyanine 5 fluorescence is performed using a confocal scanning instrument containing two tuned lasers, which excite Cyanine dyes at the appropriate wavelengths. Description Microarrays were scanned on an Agilent scanner (G2565AA) at 100% laser power, 30% PMT.Data extraction was carried out with Agilent Feature Extraction software (version 9.1), and normalization was done using linear per array algorithm according to the manufacturerâs protocol. Data processing Feature extracted data was analyzed using GeneSpring GX v 7.3.1 software from Agilent. Normalization of the data was done in GeneSpring GX using the recommended one color Per Chip and Per Gene Data Transformation: Set measurements less than 0.01 to 0.01, Per Chip: Normalize to 50th percentile, Per Gene: Normalize to Specific Samples. Further quality control of normalized data was done using correlation based condition tree to eliminate bad experiments. Differentially regulated Genes were filtered with cutoff of > 1.5 for Up regulation and < 0.55 for Down Regulation were obtained. Differentially regulated genes were clustered using gene tree to identify significant gene expression patterns.
2010-08-18 | E-GEOD-17034 | biostudies-arrayexpress
Project description:Retinoblastoma Y79 cell line was treated with scopoletin in vitro and RNA was isolated for microarray experiment to study the changes in the whole gene expression profiling in the Y79 cell line. The study identified 1675 up-regulated genes (>1.0 fold) and 1879 down-regulated genes (<-1 fold) in response to scopoletin treatment Slides:Whole genome Human 4x44k array ï Starting material: Cells in RNA Later ï RNA Samples used: Y79_c_Noni Extract,Control ï Labeling kit: Agilentâs Quick-Amp labeling Kit ï Labeling Method: T7 promoter based-linear amplification to generate labeled complementary RNA ï Total RNA and cRNA Purification Kit: Qiagenâs RNeasy minikit Cat ï Hybridization Kit: Agilentâs In situ Hybridzation kit Hybridization protocol The fragmented cRNA were mixed with 25ul of 2x GE Hybridization Buffer (Agilent). About 45ul of the resulting mixture was applied to the Microarray and hybridized at 65degC for 17 hours in an Agilent Microarray Hybridization Chamber (SureHyb: G2534A) with Hybridization Oven. After hybridization, slides were washed with Agilent Gene expression Wash Buffer I for 1 minute at room temperature followed by a 1 min wash with Agilent Gene expression Wash Buffer II for 37C. Slides were finally rinsed with Acetonitrile for cleaning up and drying. Scan protocol Laser detection of Cyanine 3 and Cyanine 5 fluorescence is performed using a confocal scanning instrument containing two tuned lasers, which excite Cyanine dyes at the appropriate wavelengths. Description Microarrays were scanned on an Agilent scanner (G2565AA) at 100% laser power, 30% PMT.Data extraction was carried out with Agilent Feature Extraction software (version 9.1), and normalization was done using linear per array algorithm according to the manufacturerâs protocol. Data processing Feature extracted data was analyzed using GeneSpring GX v 7.3.1 software from Agilent. Normalization of the data was done in GeneSpring GX using the recommended one color Per Chip and Per Gene Data Transformation: Set measurements less than 0.01 to 0.01, Per Chip: Normalize to 50th percentile, Per Gene: Normalize to Specific Samples. Further quality control of normalized data was done using correlation based condition tree to eliminate bad experiments. Differentially regulated Genes were filtered with cutoff of > 1.5 for Up regulation and < 0.55 for Down Regulation were obtained. Differentially regulated genes were clustered using gene tree to identify significant gene expression patterns.
2010-08-18 | E-GEOD-16998 | biostudies-arrayexpress
Project description:Ischemic heart disease is the leading cause of heart failure. Both clinical trials and experimental animal studies demonstrate that chronic hypoxia can induce contractile dysfunction even before substantial ventricular damage, implicating a direct role of oxygen in the regulation of cardiac contractile function. Prolyl hydroxylase domain (PHD) proteins are well recognized as oxygen sensors and mediate a wide variety of cellular events by hydroxylating a growing list of protein substrates. Both PHD2 and 3 are highly expressed in the heart, yet their functional roles in modulating contractile function remain incompletely understood. Here, we report that combined deletion of PHD2 and 3 dramatically decreases the expression of phospholamban (PLN), results in sustained activation of CaMKII and sensitizes mice to myocardial injury induced by chronic β-adrenergic stress. We provide evidence that thyroid hormone receptor-α (TR-α), a transcriptional regulator of PLN, interacts with PHD2 and 3 and is hydroxylated at two proline residues. Inhibition of PHDs increases the interaction between TR-α and nuclear receptor co-repressor 2 (NCOR2) and suppresses PLN transcription. These observations provide new mechanistic insights into how oxygen directly modulates cardiac contractility, providing the possibility that cardiac function can be modulated therapeutically by tuning PHD enzymatic activity. Two-condition experiment comparing gene expression in hearts isolated from PHD2/3f/f; Cre-/- and PHD2/3f/f; Cre+/- mice. Three biological replicates (mouse hearts) per condition.
2016-04-01 | E-GEOD-67726 | biostudies-arrayexpress
Project description:In vertebrates, the presence of viral RNA in the cytosol is sensed by members of the RIG-I like receptor (RLR) family , which signal to induce production of type I interferons (IFN). These key anti-viral cytokines act in a paracrine and autocrine manner to induce hundreds of interferon-stimulated genes (ISGs), whose protein products restrict viral entry, replication and budding. ISGs include the RLRs themselves: RIG-I, MDA5 and the least-studied family member, LGP2. In contrast, the IFN system is absent in plants and invertebrates, which defend themselves from viral intruders using RNA interference (RNAi). In RNAi, the endoribonuclease Dicer cleaves virus-derived double stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that target complementary viral RNA for cleavage. Interestingly, the RNAi machinery is conserved in mammals and we have recently demonstrated that it is able to participate in mammalian antiviral defence in conditions in which the IFN system is suppressed. In contrast, when the IFN system is active, one or more ISGs act to mask or suppress antiviral RNAi. Here, we demonstrate that LGP2 constitutes one of the ISGs that can inhibit antiviral RNAi in mammals. We identify Dicer as an LGP2-associated protein and show that LGP2 inhibits Dicer cleavage of dsRNA into siRNAs both in vitro and in vivo. Further, we show that in cells lacking an IFN response, ectopic expression of LGP2 interferes with RNAi-dependent suppression of gene expression. Thus, the inefficiency of RNAi as a mechanism of antiviral defence in mammalian somatic cells can be in part attributed to Dicer inhibition by LGP2 induced by type I IFNs.
2018-01-17 | PXD008364 | Pride
Project description:Adult male mice were exposed to 150 mg/kg benzo(a)pyrene (BaP) or solvent for 3 days and sampled 4 or 24 hours after the last dose. mRNA expression levels in adult mouse liver were measured using Agilent arrays. We found widespread changes in mRNA in pathways consistent with known response (xenobiotic metabolism, glutathione). We also found that BaP caused changes in the circadian rhythm pathway. We measured miRNA changes in the same samples and found no evidence for any change in transcription of miRNA as a result of the exposure. Keywords: Toxicology, time course Adult male B6C3F1 mice were exposed to a daily dose of corn oil (vehicle control group) or 150 mg/kg BaP (treatment group) for 3 days (n=5 per group). Mice were sacrificed at 4 h or 24 h following the last dose and liver lobes were extracted and flash frozen. RNA was extracted from median liver lobes and hybridized to the Agilent 4 x 44K mouse DNA arrays using a randomized block design.
2010-10-18 | E-GEOD-18789 | biostudies-arrayexpress
Project description:Transcriptional profile of 21-days differentiated LD Caco-2 cells compared to 21-days differentiated HD Caco-2 cells. Goal was to find some differences in the genome profile of the LD and HD Caco-2 cells that could be addressed to the different protocols used for the line maintenance. Two-condition experiment: 21-days differentiated Caco-2 cells maintained with the LD protocol and 21-days differentiated Caco-2 cells maintained with the HD protocol. Dye-swap technical replicates.
2011-04-25 | E-GEOD-21518 | biostudies-arrayexpress
Project description:Gene expression analysis study in cerulenin treated - 7µg/ml cerulenin treated Y79 cells and control Y79 cells Organism used: Homo sapiens ï Slides: Gene Expression Whole Human Genome 4x44k ï Starting material: Cells in RNA later ï RNA Samples used: :Control Y79 and Treated_Cerulenin ï Labeling kit: Agilents Low input RNA linear amplification Kit one color ï Labeling Method: T7 promoter based-linear amplification to generate labeled complementary RNA ï Total RNA and cRNA Purification Kit: Qiagenâs RNeasy minikit ï Hybridization Kit: Agilentâs In situ Hybridzation kit Hybridization protocol The fragmented cRNA were mixed with 25ul of 2x GE Hybridization Buffer (Agilent). About 45ul of the resulting mixture was applied to the Microarray and hybridized at 65degC for 17 hours in an Agilent Microarray Hybridization Chamber (SureHyb: G2534A) with Hybridization Oven. After hybridization, slides were washed with Agilent Gene expression Wash Buffer I for 1 minute at room temperature followed by a 1 min wash with Agilent Gene expression Wash Buffer II for 37C. Slides were finally rinsed with Acetonitrile for cleaning up and drying. Scan protocol Laser detection of Cyanine 3 and Cyanine 5 fluorescence is performed using a confocal scanning instrument containing two tuned lasers, which excite Cyanine dyes at the appropriate wavelengths. Description Microarrays were scanned on an Agilent scanner (G2565AA) at 100% laser power, 30% PMT.Data extraction was carried out with Agilent Feature Extraction software (version 9.1), and normalization was done using linear per array algorithm according to the manufacturerâs protocol. Data processing Feature extracted data was analyzed using GeneSpring GX v 7.3.1 software from Agilent. Normalization of the data was done in GeneSpring GX using the recommended one color Per Chip and Per Gene Data Transformation: Set measurements less than 0.01 to 0.01, Per Chip: Normalize to 50th percentile, Per Gene: Normalize to Specific Samples. Further quality control of normalized data was done using correlation based condition tree to eliminate bad experiments. Differentially regulated Genes were filtered with cutoff of > 1.5 for Up regulation and < 0.55 for Down Regulation were obtained. Differentially regulated genes were clustered using gene tree to identify significant gene expression patterns.
2010-08-18 | E-GEOD-16999 | biostudies-arrayexpress
Project description:Esophageal cancer is among the 10 most common malignancies and ranks as the 6th leading cause of death from cancer. It constitutes 7% of all gastrointestinal cancers and is one of the most lethal of all cancers. The large variation in the incidence of esophageal cancer in different geographic regions has often been thought to be due to variation in exposure to environmental factors; however, hereditary factors may also contribute to the variation in rates. A positive family history was found to be associated with an increased risk of esophageal cancer in several case-control and cohort studies in China. Familial aggregation also has been observed in Iran and Japan. The cancer data generated from six hospital based cancer registries in India under National Cancer Registry Programme (NCRP, Annual Report, 2003-2004) has revealed that the occurrence of esophageal cancer in Assam is highest in India. The aggregation of esophageal cancer in families is a long-observed and well-documented phenomenon in Assam of North-east part of India (AAR=32.6). In Assam high incidence of esophageal cancer with familial aggregation need investigation for etiology. The etiology of esophageal cancer in Northeast Indian population is different from other population at India due to wide variations in dietary habits or nutritional factors, tobacco chewing and alcohol habits. With in these high-risk regions, studies have shown a strong tendency toward familial aggregation, suggesting that genetic susceptibility, in conjunction with potential environmental exposures, may be involved in the etiology of esophageal cancer. Familial clustering of cancer has been one of the main avenues to the understanding of cancer etiology and the signal to the involvement of heritable genes. Familial clustering of cancer may be due to environmental factors shared by family members or due to shared genes. However, the familial aggregation of esophageal cancer among the population in northeast India may reflect the influence of environmental factors operating on individuals who are already genetically susceptible. Epidemiological studies indicate that tobacco smoking and alcohol consumption are the major factors for esophageal cancer, the role of genetic factors for familial aggregation has not been elucidated. In this study, tumor and matched normal tissue from esophageal squamous cell carcinoma patients with a family history of upper gastrointestinal cancer were analyzed using cDNA microarray containing 10,000genes to evaluate gene expression differences in esophageal squamous cell carcinoma patients with a family history of upper gastrointestinal cancer from a high- risk area in India. To identify alteration in genes and molecular functional pathways in esophageal cancer in a high incidence region of India where there is wide spread use of tobacco and betel quid with fermented areca nuts and familial aggregation cancer. Low RNA Input Fluorescent Linear Amplification Kit (Agilent, Santa Clara, CA) was used for labeling. Briefly, both first and second strand cDNA were synthesized by incubating 500ng of total RNA with 1.2ul of oligo dT-T7 Promoter Primer in nuclease-free water at 65 â¦C for 10 min followed by incubation with 4.0ul of 5à First strand buffer, 2ul of 0.1M DTT, 1 ul of 10mM dNTP mix, 1ul of 200 U/ul MMLV-RT, and 0.5ul of 40U/ul RNaseOUT, at 40 â¦C for 2 hour. Immediately following cDNA synthesis, the reaction mixture was incubated with 2.4 ul of 10 mM Cyanine-3-CTP or 2.4 ul of 10 mM Cyanine-5-CTP (Perkin-Elmer, Boston, MA), 20ul of 4X Transcription buffer, 8 ul of NTP mixture, 6 ul of 0.1M DTT, 0.5 ul of RNaseOUT, 0.6ul of Inorganic pyrophosphatase, 0.8 ul of T7 RNA polymerase, and 15.3ul of nuclease-free water at 40 â¦C for 2 hour. Qiagenâs RNeasy mini spin columns were used for purifying amplified aRNA samples. The quantity and specific activity of cRNA was determined by using NanoDrop ND-1000 UV-VIS Spectrophotometer version3.2.1. Samples with specific activity >8 were used for hybridization. 825ng of each Cyanine 3 or Cyanine 5 labeled cRNA in a volume of 41.8ul were combined with 11ul of 10x Blocking agent and 2.2ul of 25x Fragmentation buffer (Agilent), and incubated at 60deg C for 30 minutes in dark. The fragmented cRNA were mixed with 55ul of 2x Hybridization Buffer (Agilent). About 110ul of the resulting mixture was applied to the Microarray and hybridized at 65degC for 17 hours in an Agilent Microarray Hybridization Chamber (SureHyb: G2534A) with Hybridization Oven. After hybridization, slides were washed with Agilent Gene expression Wash Buffer I for 1 minute at room temperature followed by a 1 min wash with Agilent Gene expression Wash Buffer II for 37C. Slides were finally rinsed with Acetonitrile for cleaning up and drying. Microarrays were scanned on an Agilent scanner (G2565AA) at 100% laser power, 30% PMT.Data extraction was carried out with Agilent Feature Extraction software (version 9.1), and normalization was done using linear per array algorithm according to the manufacturerâs protocol. The data was analysed by GeneSpring GX and Biointerpreter software from Genotypic Technology, Bangalore. The differential expression was considered if the Log 2 mean of at least -1 for the down regulated genes and +1 for the upregulated genes. We considered only the genes that were reproducible from all replicates.
2010-06-25 | E-GEOD-10127 | biostudies-arrayexpress