Real-time PCR quantitation of glucocorticoid receptor alpha isoform
Contribuinte(s) |
UNIVERSIDADE DE SÃO PAULO |
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Data(s) |
26/08/2013
26/08/2013
2004
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Resumo |
Abstract Background The expression of glucocorticoid-receptor (GR) seems to be a key mechanism in the regulation of glucocorticoid (GC) sensitivity and is potentially involved in cases of GC resistance or hypersensitivity. The aim of this study is to describe a method for quantitation of GR alpha isoform (GRα) expression using real-time PCR (qrt-PCR) with analytical capabilities to monitor patients, offering standard-curve reproducibility as well as intra- and inter-assay precision. Results Standard-curves were constructed by employing standardized Jurkat cell culture procedures, both for GRα and BCR (breakpoint cluster region), as a normalizing gene. We evaluated standard-curves using five different sets of cell culture passages, RNA extraction, reverse transcription, and qrt-PCR quantification. Intra-assay precision was evaluated using 12 replicates of each gene, for 2 patients, in a single experiment. Inter-assay precision was evaluated on 8 experiments, using duplicate tests of each gene for two patients. Standard-curves were reproducible, with CV (coefficient of variation) of less than 11%, and Pearson correlation coefficients above 0,990 for most comparisons. Intra-assay and inter-assay were 2% and 7%, respectively. Conclusion This is the first method for quantitation of GRα expression with technical characteristics that permit patient monitoring, in a fast, simple and robust way. This work was supported by FAP – Fundação de Amparo à Pesquisa da Santa Casa de São Paulo. This work was supported by FAP – Fundação de Amparo à Pesquisa da Santa Casa de São Paulo. |
Identificador |
1471-2199 http://www.producao.usp.br/handle/BDPI/32816 10.1186/1471-2199-5-19 |
Idioma(s) |
eng |
Relação |
BMC Molecular Biology |
Direitos |
openAccess Melo et al; licensee BioMed Central Ltd. - This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
Tipo |
article original article |