title: Cytokine mRNA quantification by real-time PCR.
abstract: Real-time PCR represents a new methodology that accurately quantifies nucleic acids. This has been made possible by the use of fluorogenic probes, which are presented in two forms, namely hydrolysis probes (also called TaqMan probes) and hybridisation probes. We decided to apply this methodology to cytokine mRNA quantification and this led us to the development of a protocol that provides an easy way to develop and perform rapidly real-time PCR on a Lightcycler instrument. It was made possible by the use of freely available software that permits a choice of both the hydrolysis probe and the primers. We firstly demonstrated that the reproducibility of the method using hydrolysis probes compares favourably with that obtained with hybridisation probes. We then applied this technique to determine the kinetics of IL-1ra, IL-1beta, IL-5, IL-13, TNF-alpha and IFN-gamma induction upon stimulation of human peripheral blood mononuclear cells (PBMC) by phytohaemagglutinin (PHA). Finally, the method was also used successfully to demonstrate that IFN-alpha induces IL-10 mRNA accumulation in human monocytes.
authors.affiliation: Département d'Immunologie-Hématologie-Transfusion, Hôpital Erasme, Brussels, Belgium.
authors.forename: Patrick
authors.initials: P
authors.lastname: Stordeur
authors.forename: Lionel F
authors.initials: LF
authors.lastname: Poulin
authors.forename: Ligia
authors.initials: L
authors.lastname: Craciun
authors.forename: Ling
authors.initials: L
authors.lastname: Zhou
authors.forename: Liliane
authors.initials: L
authors.lastname: Schandené
authors.forename: Aurore
authors.initials: A
authors.lastname: de Lavareille
authors.forename: Stanislas
authors.initials: S
authors.lastname: Goriely
authors.forename: Michel
authors.initials: M
authors.lastname: Goldman
mesh_terms: D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D010835:Phytohemagglutinins / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis* / Q000096:biosynthesis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D016207:Cytokines; D010835:Phytohemagglutinins; D012333:RNA, Messenger