Measurement of relative quantities of different HLA-A and -B mRNAs in cells by reverse transcription-polymerase chain reaction and denaturing gradient gel electrophoresis.
In order to determine the relative quantities of different HLA-A and -B mRNAs in cells, we have developed a simple and reliable method by using reverse transcription-polymerase chain reaction (RT-PCR), denaturing gradient gel electrophoresis (DGGE) and phosphor imaging analysis. Cytoplasmic RNA from lymphoblastoid cell lines with well-characterized HLA phenotypes are reversely transcribed with a primer specific for all HLA-A and -B antigens. The first-strand cDNA is used as template for quantitative PCR. The primer pair used for quantitative PCR are specific for all class I HLA and one of the primers is labeled with [gamma-32P]ATP. The amplified sequences include parts of exon 2 and exon 3 which contain most polymorphic residues in class I HLA molecules. The RT-PCR products containing the amplified HLA-A and -B sequences are separated by DGGE. The radioactivities of different DNA bands separated in denaturing gradient polyacrylamide gels are measured by phosphor imaging and used to determine the relative amounts of HLA-A and -B mRNAs. This approach is validated by using samples containing known quantities of different HLA-A and -B mRNA transcripts and confirmed by S1 nuclease protection assay. The combined RT-PCR/DGGE approach therefore provides a simple and reliable method for quantitation of relative amounts of different HLA-A and -B mRNAs in cells. This method should also be useful for studying the expression of other highly conserved and duplicated genes.
Liu
K
K
Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville 32610, USA.
Kao
K J
KJ
D002461:Cell Line, Transformed; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D015234:HLA-A Antigens / Q000032:analysis / Q000235:genetics*; D015235:HLA-B Antigens / Q000032:analysis / Q000235:genetics*; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011489:Protein Denaturation; D012333:RNA, Messenger / Q000032:analysis*; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014158:Transcription, Genetic
D015234:HLA-A Antigens; D015235:HLA-B Antigens; D012333:RNA, Messenger; D015719:Single-Strand Specific DNA and RNA Endonucleases