Project description:We developed a novel approach to isolate tumor cells with high purity from blood which was subjected to immunomagnetic enrichment using EpCAM beads followed by fluorescence activated cell sorting (IE/FACS) to isolate EpCAM-positive cells away from leukocytes (CD45+). Duplicate samples of 20 cells were isolated from the same enriched blood from MBC patients and then subjected to DNA and RNA profiling in parallel. For DNA profiling, sorted cells were subjected to BAC array comparative genomic hybridization analysis following whole genome amplification. For RNA profiling, QPCR analysis was performed on sixty four (64) cancer-related genes using Taqman® low density arrays.
Project description:Reuse of materials in DNA hybridization based methods has been known since the advent of Southern membranes. Array based comparative genomic hybridization is essentially Southern hybridization with multiple probes immobilized on a solid surface. We have shown that comparative genomic hybridization microarrays fabricated with maskless array synthesizer technology can be used up to four times with application of 1,3-dimethylurea as array-stripping agent. We reproducibly detected chromosomal aberrations, 0.6 to 22.4 Mb in size, in four hybridization rounds using regenerated microarray slides. We have also demonstrated that regenerated arrays can detect smaller alterations, 16 – 200 kbp, such as common copy number variants, as well as complex aberration profiles in tumor.
Project description:Genome-wide copy number changes were monitored using array comparative genomic hybridization (aCGH) of laser-capture microdissected prostate cancer samples spanning stages of prostate cancer progression including precursor lesions, clinically localized disease and metastatic disease. A total of 62 specific cell populations from 38 patients were profiled. Keywords: Disease state analysis using array-based comparatavie genomic hybridization
Project description:We have used microarray comparative genomic hybridization (aCGH) at 1Mb resolution to study copy number changes in a series of WHO Grade II Astrocytomas (n=21). We have used Illumina arrays to study genome-wide expression patterns in a series of WHO Grade II Astrocytomas (n=10). Keywords: Array Comparative Genomic Hybridization (aCGH), Expression microarray
Project description:Array Comparative Genomic Hybridization (aCGH) of 70 pancreatic ductal adenocarcinoma (PDAC) samples was performed on Agilent 244K CGH arrays in order to find common genomic aberrations for cancer gene discovery. Additionally, matched expression profiling on Agilent 44K arrays was performed. Common copy number aberrations were identified in order to identify a list of putative cancer genes. Expression profiling data was used to further enrich this list of putative cancer genes for more likely candidates. Last, the most promising candidates were functionally interrogated using RNA interference-mediated knockdown to mimic loss. Well-known PDAC cancer genes were observed as amplified (KRAS and MYC) and deleted (CDKN2A, TGFBR2, SMAD4, and MAP2K4).
Project description:G-banding of human embryonic stem cells (hESC) has proved their predisposition to aneuploidy of chromosomes 12, 17 and X. Now, using array-based comparative genomic hybridization, we find that hESC also accumulate other recurrent chromosomal abnormalities, such as duplications of stemness genes, submicroscopic instability of 20q11.21 and the appearance of a derivative chromosome 18. Keywords: comparative genomic hybridization, genomic integrity of human embryonic stem cells
Project description:Parallel DNA and RNA profiling of EpCAM-positive cells in bone marrow and primary tumor tissue with positive disseminated tumor cell (DTC) count via immunomagnetic Enrichment/Flow Cytometry (IE/FC) of metastatic breast cancer (MBC) patients confirm their malignant nature We developed a novel approach to isolate tumor cells with high purity from bone marrow which was subjected to immunomagnetic enrichment using EpCAM beads followed by fluorescence activated cell sorting (IE/FACS) to isolate EpCAM-positive cells away from leukocytes (CD45+). For DNA profiling, sorted cells were subjected to BAC array comparative genomic hybridization analysis following whole genome amplification. For RNA profiling, QPCR analysis was performed on sixty four (64) cancer-related genes using Taqman® low density arrays. For non-tumor controls, RNA profiling was performed on matched leukocytes (CD45+) isolated from the same enriched bone marrow samples.
