RESUMO
Available ribonucleic acid (RNA) amplification methods are extensively tested for reproducibility, but only a few studies additionally deal with potential amplification bias. On targeted arrays, we evaluated three amplification protocols, which are less time consuming than the commonly used T7-RNA polymerase based in vitro transcription protocols and therefore may be more suitable for clinical use: Template-switching polymerase chain reaction (PCR), Ribo-single primer isothermal amplification and a random primer-based PCR. Additionally, a more sensitive labelling method, Dendrimer labelling, was evaluated. All methods were compared to unamplified RNA labelled at reverse transcription. From our results, we conclude that RNA amplification with template-switching PCR is highly reproducible and results in a reliable representation of the starting RNA population. We then assessed whether RNA amplification of clinical breast and thyroid cancer samples with template-switching PCR showed robust performance when altered cycle numbers or partially degraded RNA were used. Template-switching PCR proved to be a very reliable method for global RNA amplification, even when starting from partially degraded RNA down to a RNA Integrity Number of 4.3. In conclusion, template-switching PCR amplification promises to help micro-array expression profiling of limited amounts of human samples on its way to a clinical routine.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Carcinoma Papilar/genética , Perfilação da Expressão Gênica/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Neoplásico/genética , Neoplasias da Glândula Tireoide/genética , Bacteriófagos , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Papilar/patologia , Linhagem Celular Tumoral , RNA Polimerases Dirigidas por DNA/genética , Feminino , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase/métodos , RNA Neoplásico/análise , Reprodutibilidade dos Testes , Temperatura , Neoplasias da Glândula Tireoide/patologia , Proteínas Virais/genéticaRESUMO
Microarray studies are increasingly used for toxicological research and even for the development of new toxicological test methods. Since gene-expression changes in cultured cells can be conveniently measured with microarrays, this method might be of use for in vitro toxicity testing, for example, in the field of contact sensitization. Allergic contact dermatitis, the clinical manifestation of contact sensitization, may occur when sensitizing chemicals enter the skin and get in contact with epidermal and dermal antigen-presenting cells. The resulting maturation process in these cells can be measured by employing gene-expression analysis. Biomarkers currently known seem to be insufficient to identify all kinds of contact sensitizers, which may partly activate different signaling pathways (e.g., metal or organic sensitizers). Therefore, genome-wide screenings using whole-genome DNA microarrays and extensive data analysis can be performed in order to identify additional genes. Ultimately, marker genes detected in whole-genome experiments can be included in small-scale-targeted microarrays in order to establish the final test method.
Assuntos
Alérgenos , Dermatite Alérgica de Contato , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Alérgenos/genética , Alérgenos/imunologia , Animais , Antígenos CD/imunologia , Linhagem Celular , Células Dendríticas/citologia , Células Dendríticas/imunologia , Dermatite Alérgica de Contato/genética , Dermatite Alérgica de Contato/imunologia , Antígenos HLA/imunologia , Humanos , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
BACKGROUND: Animal tests have been used to characterize the potential of chemicals to produce allergic contact dermatitis, but this approach is increasingly a matter of public and political concern. Our aim was to develop and validate an alternative in vitro test that can identify contact allergens. METHODS: We developed a targeted microarray containing oligonucleotide probes for 66 immune-relevant genes and analyzed gene expression in monocyte-derived dendritic cells (Mo-DCs) treated with 1 irritant (SDS) and 2 prominent contact allergens, nickel and Bandrowski's base (BB), which is the oxidation product of the most important hair dye allergen, p-phenylenediamine. RESULTS: Comparing RNA amounts in chemical-treated and solvent-treated cells, we identified significant changes in the expression of 21 genes and 10 genes after exposure of immature DCs (iDCs) to nickel and BB, respectively, but not after exposure to SDS. Eight genes were differentially expressed after application of both nickel and BB. Real-time PCR was used to confirm the results for selected genes. CONCLUSION: We propose a microarray-based in vitro test that might allow the identification of contact allergens. Independently from donor variability, several immune-relevant genes were up- or downregulated after the application of the tested sensitizers to iDCs, therefore presenting potential marker genes. While reducing the number of laboratory animals used, this test would also enable reliable analysis of chemicals using a human system.