Allele-specific quantification of HLA-DQB1 gene expression by real-time reverse transcriptase-polymerase chain reaction

In addition to coding region polymorphism, allele-specific variation in the upstream regulatory region of the HLA-DQB1 gene has been detected. Reporter gene assays and transfection studies have indicated that HLA-DQB1 promoter polymorphism may be of functional significance. The aim of this study was to utilize real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for allele-specific quantification of HLA-DQB1 expression and to analyze cell-specific HLA-DQB1 expression in vivo . For the allele-specific quantification of DQB1 gene products, a real-time RT-PCR set of primer pairs ( n =27) and probes ( n =5) targeting exon 2 variability was established. The robustness and integrity of the assay system were confirmed by using recombinant DQB1 exon 2 plasmid clones as active exogenous controls. Sensitivity and reproducibility were assessed by serial dilution and allelic mixing analyses. In application to the study of allele-specific expression of DQB1 gene products during cytokine-driven maturation of monocyte-derived dendritic cells, differential patterns of allelic expression in heterozygous individuals were observed for DQB1 * 0301, compared to DQB1 * 0501 and DQB1 * 0602. At maximum, 1.9-fold ( * 0301/ * 0501) and 2.5-fold ( * 0301/ * 0602) higher induction was seen for DQB * 0301. In conclusion, HLA-DQB1 expression can be analyzed by real-time RT-PCR suitable for cell- and allele-specific detection of HLA-DQB1 transcripts in homo- and heterozygous combinations.