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.
Ferstl
B
B
Department of Medicine III, Institute for Clinical Immunology, University of Erlangen-Nuremberg, Erlangen, Germany.
Zacher
T
T
Lauer
B
B
Blagitko-Dorfs
N
N
Carl
A
A
Wassmuth
R
R
D000483:Alleles; D001483:Base Sequence; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D016207:Cytokines / Q000494:pharmacology; D003713:Dendritic Cells / Q000378:metabolism; D015870:Gene Expression; D005787:Gene Frequency / Q000235:genetics; D006683:HLA-DQ Antigens / Q000096:biosynthesis / Q000235:genetics*; D059866:HLA-DQ beta-Chains; D006579:Heterozygote; D006801:Humans; D008969:Molecular Sequence Data; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016207:Cytokines; D006683:HLA-DQ Antigens; D059866:HLA-DQ beta-Chains; C069051:HLA-DQB1 antigen