Semi-quantification of human C-C chemokine mRNAs with reverse transcription/real-time PCR using multi-specific standards

J Immunol Methods. 2000 Jul 31;241(1-2):109-19. doi: 10.1016/s0022-1759(00)00210-6.


A reverse transcription/real-time polymerase chain reaction (PCR) assay was established to semi-quantify the mRNA levels of the human C-C chemokines RANTES, MIP-1beta and MCP-1 relative to the housekeeping gene beta-actin. The assay showed a high sensitivity (below 60 cDNA molecules/10 microl reaction) and dynamic range (8 log units); both within-assay and inter-assay variability were below 0.06 log units and the accuracy was +/-0.06 log units for all four chemokines. Moreover, it is demonstrated that a multi-specific DNA fragment, which had previously been constructed for competitive PCR, can be used as a reliable external standard. This allows a direct semi-quantitative comparison of different chemokine mRNA levels and is a convenient alternative to the use of different sets of homologous external standards. The method was successfully applied to the semi-quantification of chemokines in human liver specimens and should be useful in further studies on steady state mRNA levels of C-C chemokines from low cell numbers or small tissue specimens.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chemokine CCL2 / genetics
  • Chemokine CCL4
  • Chemokine CCL5 / genetics
  • Chemokines, CC / genetics
  • Chemokines, CC / isolation & purification*
  • Hepatitis C, Chronic / genetics
  • Hepatitis C, Chronic / immunology
  • Humans
  • Liver / chemistry
  • Liver Cirrhosis, Biliary / genetics
  • Liver Cirrhosis, Biliary / immunology
  • Macrophage Inflammatory Proteins / genetics
  • RNA, Messenger / isolation & purification*
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Reverse Transcriptase Polymerase Chain Reaction / standards


  • Chemokine CCL2
  • Chemokine CCL4
  • Chemokine CCL5
  • Chemokines, CC
  • Macrophage Inflammatory Proteins
  • RNA, Messenger