Constructing polycompetitor cDNAs for quantitative PCR

Steven L. Reiner, Shichun Zheng, David B. Corry, Richard M. Locksley

Research output: Contribution to journalArticle

298 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)37-46
Number of pages10
JournalJournal of Immunological Methods
Volume165
Issue number1
DOIs
StatePublished - Sep 27 1993

Keywords

  • Cytokine
  • mRNA
  • Polymerase chain reaction
  • Quantitative

ASJC Scopus subject areas

  • Immunology
  • Biotechnology

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