Use of quantitative polymerase chain reaction to quantitate cytokine messenger RNA molecules

Sivadasan Kanangat, Alan Solomon, Barry T. Rouse

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

A quantitative polymerase chain reaction (PCR) using an internal control (Standard) RNA was developed for precise quantitation of human cytokine mRNA. The target mRNA and internal control were simultaneously reverse transcribed and co-amplified using the same set of primers. The amount of specific target mRNA is then quantitated by extrapolating against the standard curve generated with the internal standard. The internal control RNA consisted of linearly connected sequences of the 5′ primers of multiple cytokine genes followed by the complementary sequences to their 3' primers in the same order. This structure of the internal standard enables one to use the same internal standard for quantitating multiple cytokine mRNAs. Using this approach, we estimated the induced levels of cytokine mRNA, IL2, IL6, and TNF-α to be 2.8 × 106, 2.4 × 105 and 3.4 × 108 molecules per 1.0 μ g total cellular RNA of Jurkat, THP-1 and HL 60 cells, respectively. Comparable values were obtained when quantitation experiments were done on another batch of THP-1 and HL-60 total cellular RNA. The excellent sensitivity and reproducibility makes this approach a valuable one in following changes in cytokine gene expression in wide variety of conditions, both in vivo and in vitro.

Original languageEnglish (US)
Pages (from-to)1229-1236
Number of pages8
JournalMolecular Immunology
Volume29
Issue number10
DOIs
StatePublished - Jan 1 1992

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Cytokines
Polymerase Chain Reaction
Messenger RNA
RNA
HL-60 Cells
Interleukin-2
Interleukin-6
Gene Expression
Genes

All Science Journal Classification (ASJC) codes

  • Immunology
  • Molecular Biology

Cite this

Use of quantitative polymerase chain reaction to quantitate cytokine messenger RNA molecules. / Kanangat, Sivadasan; Solomon, Alan; Rouse, Barry T.

In: Molecular Immunology, Vol. 29, No. 10, 01.01.1992, p. 1229-1236.

Research output: Contribution to journalArticle

Kanangat, Sivadasan ; Solomon, Alan ; Rouse, Barry T. / Use of quantitative polymerase chain reaction to quantitate cytokine messenger RNA molecules. In: Molecular Immunology. 1992 ; Vol. 29, No. 10. pp. 1229-1236.
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