Project description:DNA copy number profiles can identify patients with a defect in BRCA1 or BRCA2. We previously showed that patients with a BRCA1 like profile benefit from intensified alkylating chemotherapy (IA, 4 cycles 5-fluorouracyl (5FU), epirubicin, cyclophosphamide (C) + 1 cycle carboplatin, thiotepa and cyclophosphamide with autologous stem cell transplantation (ASCT)). Presumably, this is because of the defect in error free homologous recombination DNA repair. Here we present an independent study IA chemotherapy (2 cycles induction, followed by ifosfamide, 12 g/m2; carboplatin, 900 mg/m2; and epirubicin, 180 mg/m2 with ASCT) versus anthracyclin-C (AC) or C-methothrexate-5FU (CMF) regimens in high risk breast cancer. DNA isolated from untreated resection specimens of 117 patients was labeled using Enzo labeling kit for array CGH and hybridized to a custom 135k Nimblegen platform.
Project description:Lymph-node (LN) metastases predict for high recurrence rates in breast cancer patients. Eradication of micro-metastatic tumor cells is the primary goal of adjuvant systemic treatment. Decisions regarding systemic treatment depend largely on primary tumor characteristics rather than on characteristics of their LN metastases. However, it remains unclear to what extent LN metastases, having already metastasized locally, resemble their primary breast tumors and as such will be eradicated by the systemic therapy chosen. In this study we investigated the genetic differences between primary breast cancers and their paired LN metastases using array comparative genomic hybridization analyses on a high resolution 720K Nimblegen platform. Thus far, no metastasis-specific genomic aberrations have been identified. We hypothesized that this is due to low-resolution platforms and lack of stratification on breast cancer subtypes (specifically, triple-negative (TN) versus luminal). Furthermore, we speculated that as TN tumours are known to be more genetically unstable, their LN metastases would show an increase in random copy number aberrations (CNAs). Therefore, we studied 10 primary TN breast tumour–LN pairs and 10 luminal pairs and found that all LN metastases clustered nearest to their matched tumour except for two. These two were explained by poor hybridization quality and, interestingly, the presence of two histological components in one tumour. We found no significantly altered CNAs between pairs in the whole group, nor when subdivided over subtypes; neither did we find a CNA increase in LN metastases compared to primary tumours within the TN subgroup, suggesting most CNAs are functional and not random. Our findings suggest a strong clonal relationship between primary breast tumours and its LN metastases and support the use of the primary tumor characteristics to guide adjuvant systemic chemotherapy in breast cancer patients, since primary tumors and their subsequent LN metastases seem remarkably similar, at least prior to treatment. The experiment contains 27 paired primary breast cancer samples with their lymph node metastases, analysed on a 135K whole genome CGH array
Project description:Exploratory study of copy number profiles of male breast cancer patients. The experiment contains 75 male breast cancer samples, analysed on a 135K whole genome CGH array
Project description:The aim of this experiment is to compare nucleases used in ribosome profiling using Drosophila melanogaster Kc167 cells and human U2OS osteosarcoma cells. Ribosome profiling was performed from nuclease digested samples using sucrose cushion centrifugation separated ribosomes.
Project description:Background: During the analysis of ripening related gene expression in tomato fruit, we observed a bias towards certain classes of messenger RNA based upon the type of buffer used in the initial step of the extraction procedure. We postulated that there was a functional association of the separated transcripts. To test this hypothesis, we carried out extractions from the same tissue sample where only the buffer varied. Transcripts were hybridised to Affymetrix oligo arrays and the data was analysed according to the predicted cellular component of the encoded proteins. Results: The use of an extraction buffer that lacked high levels of caeotropic agents resulted in a reduction of mRNAs encoding proteins that were either secreted or were predicted to be routed through the endomembrane system and hence could have been bound to the endoplasmic reticulum in polyribosomes. Extraction of the cell debris immediately following the initial buffer based extraction and subsequent micro array analysis revealed that the expected transcripts could be recovered. This demonstrated that the buffer was separating the mRNAs based on the cellular component of the encoded proteins. Analysis of selected transcripts by northern hybridisation again supported this theory. Conclusions: Some traditional buffers used for fruit RNA extraction selectively deplete for transcripts encoding proteins that are membrane-associated or secreted. This can be explained if polyribosomes that are bound to the endoplasmic reticulum (ER) are not effectively disrupted when the extraction buffer lacks either detergent or organic solvents. These findings have important implications with respect to experimental bias, as well as opportunities for message enrichment and protein characterisation. GeneChip analyses were performed to analyse the effect of using different extraction protocols on Solanum lycopersicum pericarp.
Project description:Ribosome profiling data from U2OS, HeLa and Kc167 cells under various lysis conditions and using immunoprecipitation to purifiy ribosome associated footprints. Two human cell lines (U2OS and HeLa cells) and a Drosophila melanogaster cell line (Kc167) are used to see if the 3'UTR reads are identified in each cell type. Immunoprecipitation of ribosomes is used to analyse if 3'UTR reads derive from ribosomes (are found with ribosome immunoprecipitates) and to which extent the lysis conditions contribute to the identification of the 3'UTR reads.
Project description:We performed chromatin run on and sequencing (ChRO-seq) on TeloHAEC cells before and after TNFα stimulation to map locations of RNA polymerase and quantify nascent transcription at RELA peaks.
Project description:Pseudomonas aeruginosa AA2 was repeatedly and intermittently exposed to tobramycin, ciprofloxacin or meropenem. Bacteria were grown on cryobeads submerged in liquid BHI medium. After 24 hours, the beads were washed and fresh medium with of without antibiotics added. After another 24 hours of incubation, the beads were washed, the bacteria removed from the beads, and used for inoculation of fresh beads. This was repeated to a total of up to ten cycles. Evolved lineages were then DNA-sequenced to screen for genome changes.
Project description:Burkholderia cenocepacia J2315 was repeatedly and intermittently exposed to tobramycin, ciprofloxacin or meropenem. Bacteria were grown on cryobeads submerged in liquid BHI medium. After 24 hours, the beads were washed and fresh medium with of without antibiotics added. After another 24 hours of incubation, the beads were washed, the bacteria removed from the beads, and used for inoculation of fresh beads. This was repeated to a total of up to ten cycles. Evolved lineages were then DNA-sequenced to screen for genome changes.