Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiling of M tuberculosis BCG strain during extended stationary and resuscitation phase of growth

ABSTRACT: 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. â¢ Organism: Mycobacterium tuberculosis BCG â¢ Slides: Agilentâs Mycobacterium tuberculosis custom array 4x44k (G2514F) AMADID No: 016013 â¢ Labeling kit: Ambion Message Amp II Bacteria a RNA Amplification Kit Cat# 1790 â¢ Labeling Method: T7 promoter based-linear amplification to generate labeled complementary RNA â¢ Total RNA and cRNA Purification Kit: Qiagenâs RNeasy minikit Cat#74104 â¢ Hybridization Kit: Agilentâs In situ Hybridzation kit 5184-3568 â¢ 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.

ORGANISM(S): Mycobacterium bovis BCG

SUBMITTER: Brahm Srivatsava

PROVIDER: E-GEOD-15642 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background; In the postabsorptive state, the portal-drained viscera are a major energy consumer, but a comprehensive overview of the adaptive fasting response in the liver is lacking. Hence, gene-expression profiling, pathway, network and gene-set enrichment analysis and immunohistochemistry were carried out on mouse liver after 0, 12, 24, and 72 hours of fasting. Results; Liver weight has fallen to 50% of control after three days of fasting, hepatocyte size was reduced with no apparent increase in apoptosis, while the basic liver structure and metabolic zonnation were preserved. Expression profiling and pathway analysis depicted four master processes, all with response peaking at 24 hours, and all but one decreasing towards 72h. Changes in gene expression were compatible with cellular energy deficiency, and metabolic adaptations were directed at enhanced glucose production, stimulation of lipid catabolism and ketone body synthesis, and strongly augmented ATP production. Consequently, the expression of genes involved in oxidative and endoplasmic reticulum stress response was increased. In addition, urea cycle genes were strongly upregulated during whole duration of fasting, in spite no obvious increase in amino-acid catabolism. Major physiological change upon continued fasting was restoration of glycogen reserves, but with reverse localization, demonstrating utilization of different glycogenic precursor compared to the fed controls. Conclusion; The changes in liver gene expression indicate a rapid onset of generating glucose and ketone bodies during short, and a return to near normal situation in prolonged fasting. The reason apparently lies in glycogen repletion, due to late fasting glucose production by (gut and) kidney. Experiment Overall Design: Male FVB mice (Charles River, Maastricht, The Netherlands) were housed at 20-22Â°C, 50-60% humidity, a 12 hours light/dark cycle, and food and water ad libitum. At 6 weeks of age, mice were fasted by removing chow for up to 72 hours before sacrifice (n = 6 per group). All animals were sacrificed between 9:00 and 10:00 a.m. by cervical dislocation. The liver was removed quickly, weighed, snap-frozen in liquid N2, and stored at -80Â°C. Total intestinal RNA was extracted from frozen tissue using TRIzol (Invitrogen), followed by a cleanup through a RNeasy column (Qiagen). The quality of RNA was assessed with the RNA 6000 Nano LabChipÂ® Kit in an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, USA). The 60-mer Mouse Development (22K) Oligo Microarray G4120A (Agilent) was used. Three arrays per experimental condition were used. Per microarray, 20 Î¼g mRNA, pooled from 2 livers, was reverse transcribed with Cy3-labelled dCTP (Perkin Elmer, Boston, USA), using the Agilent Fluorescent Direct Label Kit. Cy5-labeled cDNA produced from RNA pooled from livers of 6 fed animals served as the common reference across all arrays. Hybridized cDNAs were detected with Agilentâ??s dual-laser microarray slide scanner. The data were retrieved with Agilentâ??s Feature Extraction software 6.1.1.

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Gene expression profiling of M tuberculosis BCG strain during extended stationary and resuscitation phase of growth

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