Constructing polycompetitor cDNAs for quantitative PCR.
Analysis of mRNA levels using reverse transcription coupled with the polymerase chain reaction provides a powerful tool for studying cytokine regulation in cellular immunology. We report a novel method for cloning competitor cDNAs that is rapid, efficient and inexpensive. By linking multiple competitor cDNAs in tandem, polycompetitor constructs can be created that allow the use of a single reagent for individual PCR assays. Assays can be performed on minute samples of cell culture or tissue and can be reliably quantitated after routine gel electrophoresis without the use of densitometry or labeled nucleotides. The utility of this technique lies in the ability to produce a relatively inexpensive customized reagent that is simple to use and that allows for sensitive determinations of gene expression in a rapid and convenient manner. This method should allow investigators in many areas of biology to easily quantitate a broad range of important regulatory molecules.
Reiner
S L
SL
Department of Medicine, University of California, San Francisco.
Zheng
S
S
Corry
D B
DB
Locksley
R M
RM
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D016207:Cytokines; D017931:DNA Primers; D018076:DNA, Complementary / Q000138:chemical synthesis* / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger; D014407:Tumor Cells, Cultured
D016207:Cytokines; D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger