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

A reverse transcription/real-time polymerase chain reaction (PCR) assay was established to semi-quantify the mRNA levels of the human C-C chemokines R ANTES, MIP-1 beta and MCP-1 relative to the housekeeping gene beta-actin. T he assay showed a high sensitivity (below 60 cDNA molecules/10 mu l reactio n) and dynamic range (8 log units); both within-assay and inter-assay varia bility 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 DN A fragment, which had previously been constructed for competitive PCR, can be used as a reliable external standard. This allows a direct semi-quantita tive comparison of different chemokine mRNA levels and is a convenient alte rnative 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 st ate mRNA levels of C-C chemokines from low cell numbers or small tissue spe cimens. (C) 2000 Elsevier Science BN. All rights reserved.