Assessment of lymphokine profiles in activated lymphocytes by semiquantitative PCR.
Several methods are currently used to detect the expression of specific mRNAs in leukocytes. While Northern blot analysis and RNase protection assays are commonly chosen for quantitative assessment of mRNA levels, these methods require a significant quantity of RNA, making their use unfeasible when limiting numbers of cells are available. Alternatively, use of the reverse transcription-polymerase chain reaction (RT-PCR) technique allows detection of specific mRNAs even at low copy number. It is, however, difficult to establish the conditions which allow consistent semi-quantitative assessment of specific mRNA expression, using the RT-PCR method. We report here a modification of the RT-PCR technique, which has enabled us to compare lymphokine mRNA expression profiles in mixed cell populations activated either in vivo or in vitro. This modification is based on the use of standard RNAs generated by in vitro transcription of size-modified CD3 and IFN-gamma-specific PCR products subcloned into the pGEM3 plasmid. Equal amounts of standard RNAs are introduced into each sample, reverse transcribed and co-amplified with cellular mRNA to control the reproducibility and efficiency of the method. The template therefore follows the cellular RNA through all steps of the analysis, and the corresponding 32P-labelled PCR products are subsequently separated by PAGE procedure. The amount of radioactivity incorporated into lymphokine-specific bands is determined by densitometry and normalized against the density of standard bands. Under optimal PCR conditions this method is linear over a 50-fold range of dilutions. The technique is specific, reproducible and fast, allowing an analysis of lymphokine-specific mRNA profiles in samples containing 10(4)-10(6) cells.
Kramnik
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Department of Medicine, McGill University, Montreal, Quebec, Canada.
Skamene
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Radzioch
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D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D008213:Lymphocyte Activation / Q000235:genetics; D008222:Lymphokines / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009163:Mycobacterium bovis / Q000235:genetics; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D010641:Phenotype; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis; D013601:T-Lymphocytes / Q000276:immunology*
D000911:Antibodies, Monoclonal; D008222:Lymphokines; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D012313:RNA