Construction of an internal control to quantitate multiple porcine cytokine mRNAs by RT-PCR

N. R.Jayagopala Reddy, Bruce N. Wilkie, Bonnie A. Mallard

Research output: Contribution to journalArticle

34 Scopus citations

Abstract

A multiple internal control was constructed to be used as an exogenously added control in reverse transcription polymerase chain reaction (RT-PCR) for pig cytokines. It consists of 5' and 3' primer sequences in the order of β2 microglobulin (β2-m), IL-1, IL-4, IL-6, IL-8, IL-2, IL-10, TNF-α, TNF-β and IFN-γ. Construction was accomplished by overlapping and extension PCR (OE-PCR) utilizing short oligonucleotides. The primers were designed to give two products of different sizes on co-amplification of control and target RNAs by RT-PCR in a single tube. This permits analysis of message for several cytokines using a single exogenously added competitive template. Incorporated endonuclease sites allow construct modification by oligonucleotide addition.

Original languageEnglish (US)
Pages (from-to)868-875
Number of pages8
JournalBioTechniques
Volume21
Issue number5
Publication statusPublished - Nov 1 1996

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ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)

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