Determination of relative quantities of M-RNA for different HLA-A and -B antigens in cells by RT-PCR and DGGE

Abstract

Earlier study has shown that different HLA-A and -B antigens are differentially expressed in cells according to Mendelian inheritance (Human Immunol. 38:243, 1993). In order to study the molecular basis of differential expression of HLA antigens, we have developed a simple and rapid method using reverse transcription - polymerase chain reaction (RTPCR), denaturing gradient gel electrophoresis (DGGE) and phosphor imaging to measure relative quantities of mRNAs for different HLA-A and B antigens. Cytoplasmic mRNAs are reversely transcribed with a primer specific for HLA-A and -B mRNAs. The first-strand cDNAs are used as templates for quantitative PCR. The primers for PCR are specific for class I HLA and one of them is end-labeled with y-"P. The amplified sequences include a part of exon 2 and exon 3 which are the most polymorphic regions. The RT-PCR products of different HLA-A and -B sequences are separated by DGGE and quantified by phosphor imaging. This approach has been validated by studies in which different ratios of HLA-A and -B transcripts generated by in vitro transcription were used as templates for RTPCR. The HLA mRNA quantitation results obtained using this approach for EBV-transformed lymphoblastoid cell lines were also in parallel with the results of using SI nuclease protection assay. Our studies demonstrate that the RT-PCR and DGGE method offers a convenient and sensitive approach for precise quantitation of relative amounts of HLA-A and -B mRNAs in cells and can be applied for studying regulation of quantitative HLA expression in cells.