Competitive RT-PCR ELISA: a rapid, sensitive and non-radioactive method to quantitate cytokine mRNA.
We have developed a non-radioactive method to quantitate precisely levels of gene expression. This method is based on RT-PCR (reverse transcriptase-polymerase chain reaction) with an RNA competitor, followed by the covalent capture of the amplified DNA onto the wells of microtiter plates, and the quantitation of the PCR product by oligonucleotide hybridization and ELISA (enzyme-linked immunosorbent assay). The assay can reproducibly detect 1 zeptomole mRNA. The assay was successfully used to quantitate mRNA levels of the T cell derived cytokines interleukin-2, interleukin-4 and interferon-gamma in resting and stimulated human lymphocytes. Because it is performed in a microtiter ELISA format, this rapid, sensitive and non-radioactive method should facilitate measurements of gene expression, particularly in large clinical studies.
Taniguchi
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Department of Medicine, University of California, San Diego, La Jolla 92093-0663.
Kohsaka
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H
Carson
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DA
D001483:Base Sequence; D002478:Cells, Cultured; D016207:Cytokines / Q000235:genetics*; D004247:DNA / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000276:immunology
D016207:Cytokines; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D012313:RNA; D004247:DNA; D012194:RNA-Directed DNA Polymerase