Real-time PCR quantitation of glucocorticoid receptor alpha isoform

• Published in: BMC molecular biology Vol. 5; no. 1; p. 19
• Main Authors: Melo, Murilo R, Faria, Cláudia D C, Melo, Keli C, Rebouças, Nancy A, Longui, Carlos A
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 26.10.2004; BioMed Central
• Subjects: Adult; Child; Female; Humans; Jurkat Cells; Male; Methodology; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - standards; Receptors, Glucocorticoid - biosynthesis; Receptors, Glucocorticoid - genetics; Reference Standards; Reproducibility of Results; RNA, Messenger - analysis; RNA, Messenger - metabolism
• ISSN: 1471-2199; 1471-2199
• DOI: 10.1186/1471-2199-5-19

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 (GRalpha) 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. Standard-curves were constructed by employing standardized Jurkat cell culture procedures, both for GRalpha 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. This is the first method for quantitation of GRalpha expression with technical characteristics that permit patient monitoring, in a fast, simple and robust way.