Project description:A pilot program for monitoring proficiency in microarray facilities using three replicates of two different RNA sources. Participating laboratories prepared and hybridized targets from the six RNA samples using their own protocols. The entire process was repeated three times over a nine-month period and included approximately fifteen distinct laboratories in each testing round.
Project description:Gene amplification, copy-number increases of particular genes and surrounding genomic segments, promotes cancer progression and acquired therapy resistance. Thus, understanding genetic traits that confer gene amplification proficiency is important. The primary step for gene amplification is spontaneous DNA rearrangements initiated by DNA breaks. Here we show that mammalian cells became gene amplification-proficient when we knocked down Mre11/Rad50/Nbs1 (MRN) complex, a multifunctional complex that guards the genome from DNA breaks. Cells with reduced Mre11 experienced severe replication stress, with marked increases of single-stranded breaks followed by double-stranded breaks during DNA replication. Such breaks underlay for the increase in spontaneous gene amplification. Other traits associated with replication stress, such as impaired intra-S phase checkpoint and global transcriptional changes in DNA metabolism genes also contributed to gene amplification proficiency. Our results define Mre11 deficiency as a cause of replication stress and gene amplification proficiency and provide a candidate marker for aggressive cancer phenotypes.
Project description:Gene amplification, copy-number increases of particular genes and surrounding genomic segments, promotes cancer progression and acquired therapy resistance. Thus, understanding genetic traits that confer gene amplification proficiency is important. The primary step for gene amplification is spontaneous DNA rearrangements initiated by DNA breaks. Here we show that mammalian cells became gene amplification-proficient when we knocked down Mre11/Rad50/Nbs1 (MRN) complex, a multifunctional complex that guards the genome from DNA breaks. Cells with reduced Mre11 experienced severe replication stress, with marked increases of single-stranded breaks followed by double-stranded breaks during DNA replication. Such breaks underlay for the increase in spontaneous gene amplification. Other traits associated with replication stress, such as impaired intra-S phase checkpoint and global transcriptional changes in DNA metabolism genes also contributed to gene amplification proficiency. Our results define Mre11 deficiency as a cause of replication stress and gene amplification proficiency and provide a candidate marker for aggressive cancer phenotypes. We sequenced four samples: 2 control, GFP-expressing samples and 2 Mre11 knockdown cells
Project description:Parasites grown as either tachyzoites or bradyzoites for 40 hours were pulsed with 2,4-dithiouracil (DTU) for 1 hour. Following the pulse, mRNA was extracted and either used directly in microarray experiments (Abundance Arrays) or biotinylated and purified to select DTU labeled RNAs that were then used in microarray experiments (Synthesis Arrays). Two independent tachyzoite and bradyzoite preparations were made and duplicate microarrays were performed for each sample.
