RESUMO
We designed a low-density microarray carrying 132 DNA capture sequences highly specific for genes known to be differentially expressed among breast tumors and BCC lines or associated with specific tumor properties (cell-cycle alteration, proteolysis, adhesion, hormone sensitivity, etc). We analyzed gene expression in 11 BCC lines among which 6 had already been extensively studied (BT-474, Hs578T, MCF-7, MDA-MB-231, MDA-MB-453, T-47D) and 5 were still poorly characterized (Evsa-T, IBEP-1, IBEP-2, IBEP-3, KPL-1). Some data obtained were verified or extended by real-time polymerase chain reaction (real-time PCR), Northern blotting, Western blotting, immunohistochemistry and cell growth studies. Clustering analysis of the low-density microarray data allowed the sorting of BCC lines into two classes and supported a major discriminatory role for ER alpha, confirming data from previous studies. A few genes that are highly and specifically expressed in one cell line were identified, such as MGB1 (mammaglobin 1) in Evsa-T cells, and PIP (prolactin-inducible protein) in MDA-MB-453 BCC, suggesting an apocrine origin for these latter cells. Two BCC lines (IBEP-1 and IBEP-3) that had been previously characterized as ER alpha-negative, were classified by the low-density microarray among ER alpha-positive lines (MCF-7, T-47D, IBEP-2, BT-474, KPL-1) and were indeed confirmed as receptor-positive (at both mRNA and protein levels) and hormone-responsive cells. In conclusion, our results support the utility of a low-density microarray approach in cases where the cost and exhaustiveness of high-density microarrays may constitute a drawback; for instance, in obtaining a rapid phenotype evaluation in cell populations freshly isolated from breast tumors.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Biotinilação , Northern Blotting , Western Blotting , Neoplasias da Mama/patologia , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Análise por Conglomerados , DNA Complementar/metabolismo , Receptor alfa de Estrogênio/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Mamoglobina A , Proteínas de Neoplasias/metabolismo , Hibridização de Ácido Nucleico , Fenótipo , Reação em Cadeia da Polimerase , RNA/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Uteroglobina/metabolismoRESUMO
The DNA chips are arrays of DNA probes immobilized on solid support for simultaneous identification of many target DNA sequences. DNA chips applied to diagnosis aims to detect genomic DNA or RNA after PCR amplification. This review provides an overview of DNA chip technology, focusing on diagnostic applications. A comparison between high density and low density microarrays is given showing that low density chips are more suitable for routine applications due to their simplicity, good reproducibility, easy data management and low cost.
Assuntos
Infecções Bacterianas/diagnóstico , Doenças Genéticas Inatas/diagnóstico , Testes Genéticos/métodos , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Viroses/diagnóstico , Resistência Microbiana a Medicamentos , Desenho de Equipamento , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/classificação , Análise de Sequência com Séries de Oligonucleotídeos/tendênciasRESUMO
BACKGROUND: The MAGE-A genes encode antigens that are of particular interest for antitumor immunotherapy because they are strictly tumor specific and are shared by many tumors. We developed a rapid method to identify the MAGE-A genes expressed in tumors. METHODS: A low-density DNA microarray was designed to discriminate between the 12 MAGE-A cDNAs amplified by PCR with only one pair of consensus primers. The assay involved reverse transcription of total RNA with oligo(dT) primer, followed by PCR amplification and hybridization on a microarray. Amplification in the presence of Biotin-16-dUTP allowed subsequent detection of the amplicons on the microarray carrying 12 capture probes, each being specific for a MAGE-A gene. Probe-amplicon hybrids were detected by a streptavidin-based method. RESULTS: PCR conditions were optimized for low detection limits and comparable amplification efficiencies among all MAGE-A nucleotide sequences. The microarray assay was validated with a panel of 32 samples, by comparison with well-established reverse transcription-PCR assays relying on amplification with primers specific for each gene. Virtually identical results were obtained with both methods, except for MAGE-A3 and MAGE-A5. Detection of MAGE-A5 was more sensitive with the microarray assay. Detection of MAGE-A3 was hampered by the presence of MAGE-A6, which is 98% identical: the MAGE-A3 capture probe cross-hybridized with MAGE-A6 amplicons because these sequences differed by only a single base. CONCLUSIONS: This post-PCR microarray assay could be useful to evaluate MAGE expression in tumors before therapeutic vaccinations with MAGE-A gene products.
