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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

TitleDetermination of relative quantities of M-RNA for different HLA-A and -B antigens in cells by RT-PCR and DGGE
Authors
Issue Date1996
Citation
FASEB Journal, 1996, v. 10, n. 6 How to Cite?
AbstractEarlier 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 Identifierhttp://hdl.handle.net/10722/265774
ISSN
2017 Impact Factor: 5.595
2015 SCImago Journal Rankings: 2.775

 

DC FieldValueLanguage
dc.contributor.authorLiu, K.-
dc.contributor.authorKao, K. J.-
dc.date.accessioned2018-12-03T01:21:39Z-
dc.date.available2018-12-03T01:21:39Z-
dc.date.issued1996-
dc.identifier.citationFASEB Journal, 1996, v. 10, n. 6-
dc.identifier.issn0892-6638-
dc.identifier.urihttp://hdl.handle.net/10722/265774-
dc.description.abstractEarlier 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.languageeng-
dc.relation.ispartofFASEB Journal-
dc.titleDetermination of relative quantities of M-RNA for different HLA-A and -B antigens in cells by RT-PCR and DGGE-
dc.typeArticle-
dc.description.natureLink_to_subscribed_fulltext-
dc.identifier.scopuseid_2-s2.0-33749090286-
dc.identifier.volume10-
dc.identifier.issue6-
dc.identifier.spagenull-
dc.identifier.epagenull-

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