Identification of amplified and expressed genes in breast cancer by comparative hybridization onto microarrays of randomly selected cDNA clones

Abstract

Microarray analysis using sets of known human genes provides a powerful platform for identifying candidate oncogenes involved in DNA amplification events but suffers from the disadvantage that information can be gained only on genes that have been preselected for inclusion on the array. To address this issue, we have performed comparative genome hybridization (CGH) and expression analyses on microarrays of clones, randomly selected from a cDNA library, prepared from a cancer containing the DNA amplicon under investigation. Application of this approach to the BT474 breast carcinoma cell line, which contains amplicons at 20q13, 17q11-21, and 17q22-23, identified 50 amplified and expressed genes, including genes from these regions previously proposed as candidate oncogenes. When considered together with data from microarray expression profiles and Northern analyses, we were able to propose five genes as new candidate oncogenes where amplification in breast cancer cell lines was consistently associated with higher levels of RNA expression. These included the HB01 histone acetyl transferase gene at 17q22-23 and the TRAP100 gene, which encodes a thyroid hormone receptor-associated protein coactivator, at 17q11-21. The results demonstrate the utility of this microarray-based CGH approach in hunting for candidate oncogenes within DNA amplicons.

Molecular classification of synovial sarcomas, leiomyosarcomas and malignant fibrous histiocytomas by gene expression profiling

“…Information on the geneset can be found at http://www.icr.ac.uk/array/ array.html. The preparation of the microarray slides including gridding and blocking were as described in Clark et al (2002).…”

“…cDNA clones (Clark et al, 2002) were used to obtain expression profiles for 27 soft tissue sarcomas including nine synovial sarcomas, nine leiomyo-sarcomas and nine MFH tumours. We had previously established the high reliability of these microarray procedures for identifying overexpressed genes (Clark et al, 2002).…”

“…cDNA clones (Clark et al, 2002) were used to obtain expression profiles for 27 soft tissue sarcomas including nine synovial sarcomas, nine leiomyo-sarcomas and nine MFH tumours. We had previously established the high reliability of these microarray procedures for identifying overexpressed genes (Clark et al, 2002). In each experiment, Cy5-labelled sarcoma cDNA was cohybridised with Cy3-labelled reference cDNA from pooled human cell lines that served as an internal standard for the comparison of different experiments.…”

Role of E2F3 expression in modulating cellular proliferation rate in human bladder and prostate cancer cells

“…The control samples were labelled with Cy3 and the test samples with Cy5, and hybridized to CRUK Human Whole Genome-wide Array v1.0.0 (30K) cDNA microarray slides (http://www.crukdmf.icr.ac.uk/) coated with poly-L-lysine. The hybridization was carried out in triplicate essentially as described previously (Clark et al, 2002). Slides were incubated at 651C for 1 h and then transferred to 421C for 72 h. The signals were detected and visualized by GenePix 4000B scanner and software (Axon Instruments, Foster City, CA, USA), and data were filtered for quality by automated spot flagging and manual inspection, and analysed in Genespring (Silicon Genetics, Redwood City, CA, USA), using per spot and per chip intensity-dependent Lowess normalization.…”

Expression profile of genes from 12p in testicular germ cell tumors of adolescents and adults associated with i(12p) and amplification at 12p11.2–p12.1

“…The scatter plot was straightened based on the median Cy5 and Cy3 foreground intensity, using statistical locally weighted regression and smoothing of the scatter plots (Cleveland, 1979). A statistical identification of outliers was performed by the method described previously (Clark et al, 2002) to detect amplification events. The ratio of fluorescence intensities was calculated with the straightened data to assess the fold increase associated with each spot.…”