Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Human Cells Time of Replication (ToR)

ABSTRACT: Measurment of the ToR by comapring the DNA content of the S phase and the DNA content of the G1 phase, the method is based on the method used in the paper - Replication timing of the human genome by Woodfine et al. that was published in Hum. Mol. Genet. 2004. Briefly, after staining with Propidium Iodide, cells in G1 and S phase of the cell cycle are sorted by FACS sorter (ARIA or Vantage), DNA is then extracted from the fractions and differentially labeled with dUTP-Cy3 or dUTP-Cy5 using direct labeling (Agilent labeling kit). The labeled DNA is cohybridized to the array. Early replicating sequences correspond to the spots with high S/G1 ratio and late replicating sequences correspond to the spots with low S/G1 ratio. two human tissues were measured (MOLT4 and FFT-foreskin fibroblasts), 2 replicates for each sample

ORGANISM(S): Homo sapiens

SUBMITTER: Itamar Simon

PROVIDER: E-GEOD-17235 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Comparative analysis of DNA replication timing reveals conserved large-scale chromosomal architecture.

Yaffe Eitan E Farkash-Amar Shlomit S Polten Andreas A Yakhini Zohar Z Tanay Amos A Simon Itamar I

PLoS genetics 20100701 7

Recent evidence suggests that the timing of DNA replication is coordinated across megabase-scale domains in metazoan genomes, yet the importance of this aspect of genome organization is unclear. Here we show that replication timing is remarkably conserved between human and mouse, uncovering large regions that may have been governed by similar replication dynamics since these species have diverged. This conservation is both tissue-specific and independent of the genomic G+C content conservation. ...[more]

PMID: 20617169

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Project description:The changes in global gene expression in response to DNA damage may derive from either direct induction or repression by transcriptional regulation or indirectly by synchronization of cells to specific cell cycle phases, such as G1 or G2. We developed a model that successfully estimated the expression levels of >400 cell-cycle-regulated genes in normal human fibroblasts based on the proportions of cells in each phase of the cell cycle. By isolating effects on the gene expression associated with the cell cycle phase redistribution after genotoxin treatment, the direct transcriptional target genes were distinguished from genes for which expression changed secondary to cell synchronization. Application of this model to ionizing radiation (IR)-treated normal human fibroblasts identified 150 of 406 cycle-regulated genes as putative direct transcriptional targets of IR-induced DNA damage. Changes in expression of these genes after IR treatment derived from both direct transcriptional regulation and cell cycle synchronization. Experiment Overall Design: Normal human fibroblasts were synchronized and harvested at G0, G1, S G2 and M cell cycle phase, and logarithmically growing cells were treated with 1.5 Gy IR or sham, and harvested at 2, 6 or 24 h after the treatment. Total RNA was isolated using Qiagen RNeasy kit. The quality of all RNA samples was confirmed using an Agilent 2100 Bioanalyzer. Microarray analysis was then performed: briefly, 1 Âµg of sample RNA and global reference RNA (Stratagene) were converted to cDNA with reverse transcriptase then amplified using T7 RNA polymerase while labeling with either Cy3-dUTP or Cy5-dUTP (Agilentâ??s Low RNA Input Linear Amplification Kit). The quality of each labeled cRNA was evaluated using an Agilent 2100 Bioanalyzer prior to hybridization. 750ng of Cy3 and Cy5-labeled cRNA were used in the hybridization. The labeled cRNA from sham- or IR-treated samples was hybridized with the labeled global reference cRNA on an Agilent 22k human 1A array in a hybridization oven (Robbins Scientific Model 400, 1040-60-1AG) at 60oC for 17 h. Hybridization of sample RNA against reference RNA was done twice with dye swap. Following hybridization, the arrays were scanned using the Agilent DNA Microarray Scanner with SureScanÂ® Technology and microarray images were analyzed using Agilent Feature Extraction Software (v7.1). The gene expression level was presented as ratio of sample intensity against reference intensity.

Dataset Information

Human Cells Time of Replication (ToR)

Publications

Comparative analysis of DNA replication timing reveals conserved large-scale chromosomal architecture.

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