Cytogenomic profiling of breast cancer brain metastases reveals potential for repurposing targeted therapeutics
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ABSTRACT: Comparative genomic hybridization analysis for detection of copy number variation for cancer genes in breast cancer mestastatic brain tumors DNA was isolated and analyzed in a two-color experiment using Cancer CGH+SNP 180Kx4 arrays from Agilent and Agilent SureScan system: Cy5-labeled specimen DNA and Cy3-labeled reference Agilent (female) reference DNA
Project description:Comparative genomic hybridization analysis for detection of recurring gene copy number variation (CNV) among a set of lung cancer mestastatic brain tumors DNA was isolated and analyzed in a two-color experiment using Cancer CGH+SNP 180Kx4 arrays from Agilent and Agilent SureScan system: Cy5-labeled specimen DNA and Cy3-labeled Agilent characterized normal human reference DNA
Project description:We used BAF250b-/- ES cells, two independently derived clones, at 18 and 72 hr in culture and compared them with parental cell line (wild-type) at the same time in culture (two replications each). Total RNAs were extracted using Trizol (Invitrogen) according to manufacturer protocol. 2.5 ug of total RNA samples were labeled with Cy3-CTP using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent). A reference target (Cy5-CTP-labeled) was prepared from the Universal Mouse Reference RNA (Stratagene). Labeled targets were purified using an RNeasy Mini Kit (Qiagen) according to the Agilent's protocol, quantitated by a NanoDrop scanning spectrophotometer (NanoDrop Technologies), and hybridized to the NIA Mouse 44K Microarray v2.2 (whole genome 60-mer oligo; manufactured by Agilent Technologies, #014117) (Carter et al. 2005) according to the Agilent protocol (G4140-90030; Agilent 60-mer oligo microarray processing protocol - SSC Wash, v1.0). All hybridizations were carried out in the two color protocol by combining one Cy3-CTP-labeled experimental target and Cy5-CTP-labeled reference target. Microarrays were scanned on an Agilent DNA Microarray Scanner, using standard settings, including automatic PMT adjustment. Keywords: genetic modification design,time series design
Project description:Methylated genomic fragments from tumor DNA and matching normal control were enriched with the methylated-CpG island recovery assay (MIRA); amplified, labeled and hybridized on Agilent CpG island tiling oligo arrays. MIRA enriched samples from tumor were labeled with Cy5 while MIRA enriched DNA samples from normal tissue were labeled with Cy3. Keywords: Cancer
Project description:Methylated genomic fragments from tumor and matching normal DNA were enriched with the methylated-CpG island recovery assay (MIRA); amplified, labeled and hybridized on Agilent CpG island tiling oligo arrays. Samples from tumor were labeled with Cy5 while the matching normal were labeled with Cy3 so that methylation differences can be detected. Keywords: Cancer
Project description:Transcripional profiling of lymphocytes from patients with amyotrophic lateral sclerosis (ALS) (n=11) and healthy control subjects (n=11). The goal was to determine disease response expression signatures relevant of ALS pathogenesis that affect brain and spinal cord. The reference design was used: each Cy5-labeled cRNA sample from ALS patient or healthy control subject was cohybridized on Agilent-014850 Whole Human Genome Microarray 4x44K G4112F with the reference pool formed with equal amounts of Cy3-labeled cRNAs from each sample from the healthy control group.
Project description:To determine the binding targets of Tcf3 in mouse ESCs, ChIP combined with DNA microarray was carried out according to Agilent Mammalian ChIP-on-chip protocol (version 3)3. Briefly, immunoprecipitated (IP) DNA was blunted with T4 polymerase and ligated to linkers with T4 DNA ligase. For reference, 200 ng of input DNA was used. Ligated DNA was amplified with two rounds of PCR cycles. Two ug of each amplified sample were labeled with Cy5 for IP DNA, and Cy3 for input DNA using CGH labeling kit (Invitrogen). Four ug of each labeled sample was blocked with mouse Cot-1 DNA (Invitogen) and blocking agent (Agilent), and hybridized to DNA microarrays (G4490A, Agilent) for 40 h at 65oC. The microarrays were washed and stabilized with acetonitrile (Sigma), followed by stabilization and drying solution (Agilent). The arrays were scanned with Agilent DNA microarray scanner (G2565BA), and probe features were extracted using Feature Extraction Software (G2567AA, version 9.1). The data was then analysed with ChIP Analytics 1.3 (G4477AA), according to the Whitehead Neighbourhood Model, P(Xbar)<0.001, with intra-array median normalization 3. Bound targets which was determined by the algorithm was validated by qPCR on a selected number of genes. The false positive rate was determined. Keywords: ChIP-chip
Project description:Gene Expression Profiling of Severed Rat Medial Collateral Ligament at 1, 2, 4, 7,1 0, and 14 days Following Injury with Control and Cultured Ligament Fibroblasts and Rat Universal Reference RNA The aim of this study was to assess the genes involved in the repair of the the dense connective tissue of the a rat ligament in order to provide targets for improvement in healing. Rat whole genome microarrays (Agilent) were used in this study and Cy3 and Cy5 labeled total RNA was extracted and labeled with Cy3 or Cy5 prior to fragmentation and hybridization. Keywords: Time course of changes in gene expression in the healing of the rat medial collateral ligament.
Project description:To determine the binding targets of SMAD1/5, SMAD2 and SMAD4 in mouse ESCs, ChIP combined with DNA microarray was carried out according to Agilent Mammalian ChIP-on-chip protocol. Briefly, immunoprecipitated (IP) DNA was blunted with T4 polymerase and ligated to linkers with T4 DNA ligase. For reference, 200 ng of input DNA was used. Ligated DNA was amplified with two rounds of PCR cycles. Two ug of each amplified sample were labeled with Cy5 for IP DNA, and Cy3 for input DNA using CGH labeling kit (Invitrogen). Four ug of each labeled sample was blocked with mouse Cot-1 DNA (Invitogen) and blocking agent (Agilent), and hybridized to DNA microarrays (G4490A, Agilent) for 40 h at 65℃. The microarrays were washed and stabilized with acetonitrile (Sigma), followed by stabilization and drying solution (Agilent). The arrays were scanned with Agilent DNA microarray scanner (G2565BA), and probe features were extracted using Feature Extraction Software (version 10.0). The raw data were processed by the R package limma and the log-ratio of IP over WCE was normalized within arrays using ‘loess’ method. And the normalized log-ratios were then used for SMAD target selection. Bound targets which was determined by the algorithm was validated by ChIP-stie specific PCR on a selected number of genes. The false positive rate was determined. Keywords: ChIP-chip
Project description:Array CGH of H. pylori strain BCS100 after challenge to a new host (patients and experiment described in Graham et al., 2004, Gut, 53:1235-1243). Genomic DNA (500 ng) from the ouput from singel colony isolates obtained 15 or 90 days post innoculation were labeled with Cy5 (red) and cohybridized with 500 ng of genomic DNA from the innoculating strain BCS100 labeled with Cy3 (green). A reference experiement design type is where all samples are compared to a common reference. Keywords: reference_design
Project description:Characterization of genomic copy number changes in breast cancer cell lines BT474, SKBR3, KPL4 and MCF7 compared to a normal human female genomic DNA reference. The experiment utilized four breast cancer cell lines; BT474, SKBR3, MCF7 and KPL4. All cell lines were grown in accordance with the distributor’s instructions. All samples were hybridized once on 1M Agilent Human Genome CGH microarrays according to manufacturers instructions. Genomic DNA pooled from healthy female donors was used as a reference in all hybridizations. DNA from cell line samples were labeled with Cy5 and DNA from reference were labeled with Cy3.