Real-time PCR quantitation of glucocorticoid receptor alpha isoform.
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 (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.


RESULTS
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.


CONCLUSIONS
This is the first method for quantitation of GRalpha expression with technical characteristics that permit patient monitoring, in a fast, simple and robust way.
Melo
Murilo R
MR
Pediatric Endocrinology Unit, Irmandade da Santa Casa de Misericórdia de São Paulo, Brazil. murilomelo@uol.com.br
Faria
Cláudia D C
CD
Melo
Keli C
KC
Rebouças
Nancy A
NA
Longui
Carlos A
CA
D000328:Adult; D002648:Child; D005260:Female; D006801:Humans; D019169:Jurkat Cells; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000096:biosynthesis / Q000235:genetics*; D012015:Reference Standards; D015203:Reproducibility of Results
D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; C411470:glucocorticoid receptor alpha