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Article: Determination of relative quantities of M-RNA for different HLA-A and -B antigens in cells by RT-PCR and DGGE
Title | Determination of relative quantities of M-RNA for different HLA-A and -B antigens in cells by RT-PCR and DGGE |
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Authors | |
Issue Date | 1996 |
Citation | FASEB Journal, 1996, v. 10, n. 6 How to Cite? |
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. |
Persistent Identifier | http://hdl.handle.net/10722/265774 |
ISSN | 2023 Impact Factor: 4.4 2023 SCImago Journal Rankings: 1.412 |
DC Field | Value | Language |
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dc.contributor.author | Liu, K. | - |
dc.contributor.author | Kao, K. J. | - |
dc.date.accessioned | 2018-12-03T01:21:39Z | - |
dc.date.available | 2018-12-03T01:21:39Z | - |
dc.date.issued | 1996 | - |
dc.identifier.citation | FASEB Journal, 1996, v. 10, n. 6 | - |
dc.identifier.issn | 0892-6638 | - |
dc.identifier.uri | http://hdl.handle.net/10722/265774 | - |
dc.description.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. | - |
dc.language | eng | - |
dc.relation.ispartof | FASEB Journal | - |
dc.title | Determination of relative quantities of M-RNA for different HLA-A and -B antigens in cells by RT-PCR and DGGE | - |
dc.type | Article | - |
dc.description.nature | link_to_subscribed_fulltext | - |
dc.identifier.scopus | eid_2-s2.0-33749090286 | - |
dc.identifier.volume | 10 | - |
dc.identifier.issue | 6 | - |
dc.identifier.spage | null | - |
dc.identifier.epage | null | - |
dc.identifier.issnl | 0892-6638 | - |