Unknown,Transcriptomics,Genomics,Proteomics

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Expression Profiles of Cloned Channel Catfish (Ictalurus punctatus) Lymphoid Cell Lines and a Mixed Lymphocyte Culture

ABSTRACT: Clonal channel catfish lymphoid cell lines and mixed lymphocyte cultures (MLC) have proven extremely useful in examining immune responses at the cellular and molecular levels. To date catfish cell cultures have been biologically and phenotypically characterized using a variety of techniques including reverse transcription PCR (RT-PCR), as well as Northern and Southern Blotting. To expand the molecular characterization of these cultures, microarray analysis was employed. Clonal B (3B11), macrophage (42TA), and cytotoxic T cell (TS32.15 and TS32.17) lines and MLC were examined using a cDNA array containing ~ 2500 probes derived from EST libraries prepared from the 42TA macrophage cell line, a MLC, and 5 - 14 day old catfish fry. Analysis showed that each cell line displayed a unique RNA expression profile that included a variety of immune-related genes. Pearson correlation analysis indicated that one cytotoxic T cell line clustered with MLC, whereas a second cytotoxic T cell line was more closely associated with B cells and macrophages The microarray experiments were performed as two biological replicates and two technical replicates. In each case, gene expression in one of the three cell lines (TS32.15, TS32.17, and 42TA) or MLCs was compared against gene expression in the cell line 3B11. Biological replicates were performed on samples that were obtained from two different cell stocks that were frozen at different times and after a different number of subcultures. Technical replicates were performed as dye swaps where the dye was swapped between the test and the control cDNA. If a biological replicate was used, the control cell line also was a biological replicate.

ORGANISM(S): Ictalurus punctatus

SUBMITTER: Sai Majji

PROVIDER: E-GEOD-11920 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: skeletal muscle is a complex, versatile tissue composed of a variety of functionally diverse fiber types. Although the biochemical, structural and functional properties of myofibers have been the subject of intense investigation for the last decades, understanding molecular processes regulating fiber type diversity is still complicated by the heterogeneity of cell types present in the whole muscle organ. Methodology/Principal Findings: we have produced a first catalogue of genes expressed in mouse slow-oxidative (type 1) and fast-glycolytic (type 2B) fibers through transcriptome analysis at the single fiber level (microgenomics). Individual fibers were obtained from murine soleus and EDL muscles and initially classified by myosin heavy chain isoform content. Gene expression profiling on high density DNA oligonucleotide microarrays showed that both qualitative and quantitative improvements were achieved, compared to results with standard muscle homogenate. First, myofiber profiles were virtually free from non-muscle transcriptional activity. Second, thousands of muscle-specific genes were identified, leading to a better definition of gene signatures in the two fiber types as well as the detection of metabolic and signaling pathways that are differentially activated in specific fiber types. Several regulatory proteins showed preferential expression in slow myofibers. Discriminant analysis revealed novel genes that could be useful for fiber type functional classification. Conclusions/Significance: as gene expression analyses at the single fiber level significantly increased the resolution power, this innovative approach would allow a better understanding of the adaptive transcriptomic transitions occurring in myofibers under physiological and pathological conditions. EDL and soleus muscles were incubated with type I collagenase to dissociate intact myofibres that were separated under stereo microscope from the bulk of hyper contracted fibres. Isolated myofibres were divided in two parts: one was immersed in Laemmli buffer for fibre typing; the other was placed in RNA extraction buffer for RNA amplification. We analyzed the transcription profiles of 10 biological replicas of type 2B single muscle fibres from EDL and 10 biological replicas of type 1 single muscle fibres from soleus. Microarray competitive hybridizations were carried out against an artificial control with a balanced composition of type 1 and type 2B fibres (20 hybridizations). Each oligonucleotide is spotted in two replicates on the glass slide, so for every data two intra-slide replicas are present. The control was created as follows: three couples of soleus and EDL muscles were removed from 3 different mice and treated with collagenase. Total RNA was extracted separately from EDL and soleus muscles. By mixing about 1/3 RNA from EDL and 2/3 RNA from soleus muscles the control had approximately the same contributions of type 1 and type 2B fibres. Purified RNA samples from single fibres and from control were amplified twice using the Amino Allyl MessageAmpM-bM-^DM-" II aRNA Amplification Kit (Ambion).

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Expression Profiles of Cloned Channel Catfish (Ictalurus punctatus) Lymphoid Cell Lines and a Mixed Lymphocyte Culture

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