RESUMO
cDNA representational difference analysis (cDNA RDA) is a PCR-based subtractive enrichment procedure for the cloning of differentially expressed genes. In this study, we have further developed the procedure to take advantage of solid-phase technology, and to facilitate the use of RDA when starting material is limited. Several parameters of the PCR-based generation of cDNA representations were investigated, and a solid-phase based purification step was introduced to simplify removal of digested adapter-ends and uncleaved fragments. The use of magnetic particles increased the speed of the method, and also eliminated the risk of carry-over contamination between iterative steps of subtraction and PCR amplification. The modified protocol was evaluated in monitoring differences in gene expression in (i) a rat system consisting of livers with and without growth hormone treatment, and in (ii) a human system consisting of normal colon and colon cancer.
Assuntos
DNA Complementar/química , DNA de Neoplasias/química , Reação em Cadeia da Polimerase/métodos , Resinas Acrílicas/química , Animais , Biópsia , Clonagem Molecular , Neoplasias do Colo/genética , Primers do DNA/química , DNA Complementar/metabolismo , Eletroforese em Gel de Ágar , Hormônio do Crescimento/metabolismo , Hormônio do Crescimento/farmacologia , Humanos , Fígado/efeitos dos fármacos , Masculino , Hibridização de Ácido Nucleico/métodos , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ratos , Técnica de Subtração , Extratos de TecidosRESUMO
T cells are essential in the initiation and maintenance of immune responses. Specific interaction between T cells and a presumptive antigen occurs through recognition of an MHC-peptide complex by the T-cell receptor (TCR). The complementarity-determining region (CDR) 3 of the TCR has direct contact with the peptide. Here we describe CDR3 length variability of six different TCRBV gene families of CD4+ and CD8+ umbilical cord (UC) and peripheral blood (PB) T cells. Amplified products spanning the TCR CDR3 regions from CD4+ PB, CD4+ UC and CD8+ UC blood T cells typically displayed Gaussian-like distributions. In contrast, profound and frequent perturbations were recorded in CD8+ PB lymphocytes, with a non-Gaussian pattern in more than half of the samples studied. A substantial portion of the perturbed CD8+ subsets were clonal or oligoclonal, as determined by CDR3-length restriction, TCRBJ gene usage and nucleotide sequencing. This implies that the conditions for shaping and maintenance of the peripheral TCR repertoire are profoundly different for CD8+ and CD4+ T cells.
