Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human distinct types of fibrocyte differentiate in the presence and abscence of serum

ABSTRACT: Fibroblasts, Serum-free Fibrocytes, Serum-containing Fibrocytes, Monocytes, Macrophages, Osteoclasts, immature Dendritic Cells, and mature Dendritic Cells were all generated from 3 biological replicates from each of 3 separate donors. RNA was extracted (Ambion RNAqueous), labelled with cy3, mixed with cy5 labelled human reference (Stratagene), and hybridised to slides printed with Human AROS v4.0 oligonucleotides (Operon). Slides were scanned using a Perkin Elmer GX plus, and the data then normalised with GEPAS v4.0 and collated. Final data analysis was carried out using TMEV 4.0. SAM was performed using a 0.1% FDR. HCL and PCA were plotted from this list, and interrogation carried out using DAVID to determine pathway enrichment. After initial analysis, the Fibroblast cell type was found to significantly bias the data. Due to this, these samples were removed from the analysis, and the data re-normalised. This second data file will be submitted separately.

ORGANISM(S): Homo sapiens

SUBMITTER: Stephen Kissane

PROVIDER: E-MEXP-2434 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The screen for genes involved in the spontaneous loss of heterozygosity mutagenesis (SLM) in diploid cells was done on the collections of deletion strains of Saccharomyces cerevisiae created by the Saccharomyces Genome Deletion Project (http://www-sequence.stanford.edu/group/yeast_deletion_project/). Screens were performed on the pools of clones. Barcode microarrays were used to detect the increased SLM frequency leading to relative increase in abundance of deletion clone in a pool. Collections used were: Homozygous Diploid (HD) and Essential (E) mixed to create single HDE pool. Three mutagenesis markers were used: (1) naturally existing mating type locus, and two newly introduced markers, (2) CAN1/can1delta, (3) URA3/ura3delta. 1. To detect the increased SLM frequency at the mating type locus HDE pool was crossed to MATa or MATalpha strain. Deletion clones that gained mating competence due to loss of heterozygosity (LOH) at the mating type locus gave rise to clones with the ability to grow on minimal medium. The relative abundance of these clones was compared to the abundance in the original HDE pool. 2. To detect the increased SLM frequency at the CAN1/can1delta locus the derivative HDE pool was created. Every cell of this pool had one of two copies of CAN1 gene deleted. The increased SLM frequency led to increased relative abundance of respective deletion clone under canavanine selection and could be estimated by comparing it to the abundance when pool was grown without selection. 3. To detect the increased SLM frequency at the URA3/ura3delta locus the derivative HDE pool was created. Every cell of this pool had one of two copies of URA3 gene deleted. The increased SLM frequency led to increased relative abundance of respective deletion clone under 5M-^R-fluoroorotic acid selection and could be estimated by comparing it to the abundance when pool was grown without selection. 4. For screens of SLM at CAN1 and URA3 markers the resistance control experiments were performed to detect genes whose deletion is sufficient to enable yeast cells with functional CAN1 or URA3 to grow in the presence of canavanine or 5M-^R- fluoroorotic acid respectively. 5. To include in the analysis slow-growing deletion clones control hybridizations were performed to detect genes whose deletion extends the doubling time of yeast cells.

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Transcription profiling of human distinct types of fibrocyte differentiate in the presence and abscence of serum

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