Analysis of spontaneous mRNA cytokine production in peripheral blood.
The quantification of the cytokine mRNAs synthesised by peripheral blood cells should make it possible to estimate a "peripheral immune statute". However, an accurate quantification can only be performed from a fresh whole blood sample in which mRNA is protected against nuclease digestion, and where gene transcription is inhibited. As discussed in this note, this has been made possible by the use of surfactant reagents such as tetradecyltrimethylammonium oxalate. We performed RT-PCR for the quantification of IL-10 and IFN-gamma mRNAs spontaneously produced in peripheral blood. The results showed pronounced higher IFN-gamma transcript levels in whole blood compared to peripheral blood mononuclear cells (PBMC) from the same individuals, while no significant difference was observed for IL-10 mRNA. The higher amounts of IFN-gamma mRNA observed in blood can be attributed at least to mRNA degradation. Using a real time PCR technique, we indeed demonstrated that blood IFN-gamma mRNA is rapidly degraded in vitro, the t 1/2 being worth approximately one hour at room temperature.
Stordeur
Patrick
P
Département d'Immunologie-Hématologie-Transfusion, Hôpital Erasme, Université Libre de Bruxelles, Route de Lennik 808, 1070 Brussels, Belgium. patrick.stordeur@ulb.ac.be
Zhou
Ling
L
Goldman
Michel
M
D016207:Cytokines / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000235:genetics; D016753:Interleukin-10 / Q000235:genetics; D000644:Quaternary Ammonium Compounds; D020871:RNA Stability; D012333:RNA, Messenger / Q000097:blood* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013501:Surface-Active Agents; D050337:Trimethyl Ammonium Compounds
D016207:Cytokines; D000644:Quaternary Ammonium Compounds; D012333:RNA, Messenger; D013501:Surface-Active Agents; D050337:Trimethyl Ammonium Compounds; D016753:Interleukin-10; D007371:Interferon-gamma; C009410:tetradecyltrimethylammonium