Real-time PCR quantitation of glucocorticoid receptor alpha isoform

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.This is the first method for quantitation of GRα expression with technical characteristics that permit patient monitoring, in a fast, simple and robust way.Glucocorticoids (GC) are a vital class of steroidal hormones that mediate profound and diverse physiological effects in vertebrates. GC are key hormones in the regulation of glucose homeostasis, but other essential functions are assigned to GC as well, such as regulatory roles in development and other metabolic pathways, stress and immune responses, neurobiology, and programmed cell death. In addition, corticosteroids are among the most widely prescribed class of drugs, primarily for their anti-inflammatory and immunosuppressive roles. They are also used in many chemotherapy regimens for leukemias and other cancers due to their critical capability to induce apoptosis.Cortisol and its synthetic derivatives act upon the glucocorticoid receptor (GR), a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors. Prior to ligand binding, GR is primarily localized within the cytoplasm as an oligomeric complex composed of one receptor polypeptide, two molecules of heat shock protein 90 (hsp90), one molecule each of hsp 70, hsp 56 (immunophilin) and hsp23.