Application of differential display to immunological research.
The majority of immunological processes are mediated by cell-to-cell contact or receptor-ligand interactions that transmit intracellular signals and affect the regulation of transcription in the nucleus. As a consequence, precursor cells develop into their respective lineages and cells differentiate further during an immune response. In order to study changes in normal cells or even cells that have been isolated from diseased tissue, a number of approaches have been developed. One such method, differential display (DDRT-PCR), is a versatile technique for the analysis of gene expression that is based on RT-PCR and denaturing polyacrylamide gel electrophoresis. This technique is applicable to multiple samples of clonal or purified cell populations as well as to complex tissues and can be used to provide mRNA fingerprints. However, the main purpose of DDRT-PCR is to isolate differentially regulated genes in biological systems. The method is carried out without prior hypothesis as to which genes should be examined and so increases the possibility of identifying completely novel and unexpected changes in transcription. A major drawback has been the isolation of false positive clones and the need to confirm the results of analysis by another method. This makes DDRT-PCR labour intensive. A number of strategies have been recommended to reduce these problems, including reverse-northern analysis as a confirmatory step for screening putative differentials. In order to reduce the number of gel fingerprints that would be required to cover all the mRNAs in a cell, several focused approaches have been suggested. These include targeted differential display for the isolation of multigene families that have conserved protein domains or gene signatures and subtractive differential display whereby one population is subtracted from the other prior to screening. The purpose of this review is to provide some guidance to the immunologist who might wish to apply DDRT-PCR in their research. A number of examples where DDRT-PCR has been used successfully in immunological research are included.
Molecular Medicine Unit, University of Leeds, Clinical Sciences Building, St. James's Hospital, LS9 7TF, Leeds, UK. medma@leeds.ac.uk
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Ali
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Markham
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Isaacs
D000818:Animals; D017209:Apoptosis; D002454:Cell Differentiation; D005189:False Positive Reactions; D020869:Gene Expression Profiling / Q000379:methods*; D006098:Granulocytes / Q000166:cytology; D006801:Humans; D007113:Immunity, Innate / Q000235:genetics; D007158:Immunologic Techniques; D008214:Lymphocytes / Q000166:cytology / Q000276:immunology; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012333:RNA, Messenger