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 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.
Dumoulin
F L
FL
Department of Medicine I, University of Bonn, Germany. dumoulin@uni-bonn.de
Nischalke
H D
HD
Leifeld
L
L
von dem Bussche
A
A
Rockstroh
J K
JK
Sauerbruch
T
T
Spengler
U
U
D018932:Chemokine CCL2 / Q000235:genetics; D054407:Chemokine CCL4; D018946:Chemokine CCL5 / Q000235:genetics; D019742:Chemokines, CC / Q000235:genetics / Q000302:isolation & purification*; D019698:Hepatitis C, Chronic / Q000235:genetics / Q000276:immunology; D006801:Humans; D008099:Liver / Q000737:chemistry; D008105:Liver Cirrhosis, Biliary / Q000235:genetics / Q000276:immunology; D019402:Macrophage Inflammatory Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards
D018932:Chemokine CCL2; D054407:Chemokine CCL4; D018946:Chemokine CCL5; D019742:Chemokines, CC; D019402:Macrophage Inflammatory Proteins; D012333:RNA, Messenger