Unknown,Transcriptomics,Genomics,Proteomics

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Global gene expression in catfish spleen after injection of lipopolysaccharide

ABSTRACT: A high-density oligonucleotide microarray for channel catfish (Ictalurus punctatus) was designed and produced with Maskless Array Synthesizer technology (MAS). The microarray contained ~379,652 24-mer oligonucleotides covering ~18,999 catfish unique sequences. The global expression profiling of the catfish spleens stimulated by lipopolysaccharide (LPS) for 2h, 4h, 8h and 24h was investigated with the microarray. In the spleen samples, 409 genes were identified to be induced or repressed greater than 2-fold by LPS treatment. Self-organizing maps (SOM) clustering analysis was applied to interpret gene expression patterns, and 84 of these genes were clustered into six expression patterns. Real-time RT-PCR was used to verify the microarray results for 9 selected genes representing different expression levels. The results from real-time RT-PCR were positively correlated (R^2 = 0.87) with the results from the microarray. Channel catfish were injected with LPS or PBS carrier. Fish were killed by anesthesia overdose at 2, 4, 8, or 24h post-injection and total RNA was isolated from spleen. The experiment contained two replicate fish per time point. Relative signal intensities for each feature were generated using the Robust Multi-Array Average algorithm and log2 transformed. Transformed data were then processed using quantile normalization, and the average and std. error for each gene were calculated from the 10 perfectly matched oligos only. After detransformation, the ratio (fold change) was calculated using PBS treated samples (control) as the baseline against LPS-treated samples.

ORGANISM(S): Ictalurus punctatus

SUBMITTER: Geoffrey Waldbieser

PROVIDER: E-GEOD-3261 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Production and utilization of a high-density oligonucleotide microarray in channel catfish, Ictalurus punctatus.

Li Robert W RW Waldbieser Geoffrey C GC

BMC genomics 20060601

<h4>Background</h4>Functional analysis of the catfish genome will be useful for the identification of genes controlling traits of economic importance, especially innate disease resistance. However, this species lacks a platform for global gene expression profiling, so we designed a first generation high-density oligonucleotide microarray platform based on channel catfish EST sequences. This platform was used to profile gene expression in catfish spleens 2 h, 4 h, 8 h and 24 h after injection of ...[more]

PMID: 16740160

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Project description:With the aim of shedding light on the protection conferred by the DNA vaccines based in the G glycoprotein of viral haemorrhagic septicaemia virus (VHSV) in turbot (Scophthalmus maximus) we have used a specific microarray highly enriched in antiviral sequences to carry out the transcriptomic study associated to VHSV DNA vaccination/infection. The differential gene expression pattern in response to empty plasmid (pMCV1.4) and DNA vaccine (pMCV1.4-G860) intramuscular administration with regard to non-stimulated turbot was analyzed in head kidney at 8, 24 and 72 hours post-vaccination. Moreover, the effect of VHSV infection one month after immunization was also analyzed in vaccinated and non-vaccinated fish at the same time points. A total number of 204 juvenile turbot were divided into 3 groups, two of them containing 72 fish and the last one 60 fish. Turbot were anaesthetized by immersion in 50 mg/ml buffered tricaine methanesulfonate (MS-222; Sigma) and then, fish from the first two groups were intramuscularly (i.m.) injected with 50 µl of PBS containing 2 µg of pMCV1.4 or pMCV1.4-G860. Turbot from the last batch were i.m. inoculated with 50 µl of PBS. At 8, 24 and 72 h after injection, 12 fish were removed from the first two tanks and, at 8 h after PBS inoculation, other 12 fish were taken from the last tank. These turbot were sacrificed by anaesthetic overdose and the head kidney was removed. Equal amounts of tissue from three fish belonging to the same tank and sampling point were pooled, obtaining 4 biological replicates for each treatment and time point (3 turbot/replicate). The remaining fish (36 in the plasmid-injected groups and 48 in the PBS-inoculated tank) were maintained during one month and then, 12 fish from the PBS injected group were separated to another tank. This new group of fish was intraperitoneally (i.p.) injected with 50 µl of MEM + penicillin and streptomycin + 2% FBS (PBS - MEM group), whereas the other turbot were i.p. infected with a dose of VHSV860 of 5 x 105 TCID50/fish (pMCV1.4 - VHSV and pMCV1.4-G860 - VHSV groups). At 8, 24 and 72 hours after infection, 12 fish were removed from the VHSV-infected tanks, and at 8 h after MEM injection the 12 fish were taken from the non-infected tank. The fish were sacrificed by anaesthetic overdose and the head kidney was removed. Equal amounts of tissue from three fish belonging to the same tank and sampling point were pooled, obtaining 4 biological replicates for treatment and time point (3 turbot/replicate)

Dataset Information

Global gene expression in catfish spleen after injection of lipopolysaccharide

Publications

Production and utilization of a high-density oligonucleotide microarray in channel catfish, Ictalurus punctatus.

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