Quantification of Murine Cytokine mRNAs Using Real Time Quantitative Reverse Transcriptase PCR

Cytokine. 1999 Apr;11(4):305-12. doi: 10.1006/cyto.1998.0426.

Abstract

Recently, a novel technique for "real time" quantitative Reverse Transcriptase-PCR which measures PCR-product accumulation during the exponential phase of the PCR reaction using a dual-labelled fluorogenic probe, has been developed. This method allows direct detection of PCR-product formation by measuring the increase in fluorescent emission continuously during the PCR reaction. Here we present data validating this PCR-method for the quantification of murine cytokines and other factors playing a role in immune regulation (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p40, IL-13, IL-15, IFN-gammaTNF-alphaTGF-beta and iNOS). For each substance of interest, a set of primers and internal probe was designed, which specifically amplify the target cDNA, not co-amplifying contaminating genomic DNA. Furthermore, a corresponding reference plasmid cDNA clone was constructed, allowing direct quantification. Additionally, normalization to the housekeeping genes beta-actin or GAPDH was performed. The assay is very sensitive and accurate. It is a "closed-tube" PCR reaction, avoiding time-consuming and hazardous post-PCR manipulations and decreasing the potential risk of PCR contamination.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Cytokines / genetics*
  • DNA Primers / genetics
  • DNA Probes / genetics
  • DNA, Complementary / genetics
  • DNA, Complementary / standards
  • Evaluation Studies as Topic
  • Fluorescent Dyes
  • Mice
  • Quality Control
  • RNA, Messenger / analysis*
  • RNA, Messenger / genetics*
  • Reference Standards
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Reverse Transcriptase Polymerase Chain Reaction / standards
  • Reverse Transcriptase Polymerase Chain Reaction / statistics & numerical data
  • Sensitivity and Specificity

Substances

  • Cytokines
  • DNA Primers
  • DNA Probes
  • DNA, Complementary
  • Fluorescent Dyes
  • RNA, Messenger