CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use.
BACKGROUND
Real-time PCR is becoming a common tool for detecting and quantifying expression profiling of selected genes. Cytokines mRNA quantification is widely used in immunological research to dissect the early steps of immune responses or pathophysiological pathways. It is also growing to be of clinical relevancy to immuno-monitoring and evaluation of the disease status of patients. The techniques currently used for "absolute quantification" of cytokine mRNA are based on a DNA standard curve and do not take into account the critical impact of RT efficiency.


RESULTS
To overcome this pitfall, we designed a strategy using external RNA as standard in the RT-PCR. Use of synthetic RNA standards, by comparison with the corresponding DNA standard, showed significant variations in the yield of retro-transcription depending the target amplified and the experiment. We then developed primers to be used under one single experimental condition for the specific amplification of human IL-1beta, IL-4, IL-10, IL-12p40, IL-13, IL-15, IL-18, IFN-gamma, MIF, TGF-beta1 and TNF-alpha mRNA. We showed that the beta-2 microglobulin (beta2-MG) gene was suitable for data normalisation since the level of beta2-MG transcripts in naive PBMC varied less than 5 times between individuals and was not affected by LPS or PHA stimulation. The technique, we named CyProQuant-PCR (Cytokine Profiling Quantitative PCR) was validated using a kinetic measurement of cytokine transcripts under in vitro stimulation of human PBMC by lipopolysaccharide (LPS) or Staphylococcus aureus strain Cowan (SAC). Results obtained show that CyProQuant-PCR is powerful enough to precociously detect slight cytokine induction. Finally, having demonstrated the reproducibility of the method, it was applied to malaria patients and asymptomatic controls for the quantification of TGF-beta1 transcripts and showed an increased capacity of cells from malaria patients to accumulate TGF-beta1 mRNA in response to LPS.


CONCLUSIONS
The real-time RT-PCR technique based on a RNA standard curve, CyProQuant-PCR, outlined here, allows for a genuine absolute quantification and a simultaneous analysis of a large panel of human cytokine mRNA. It represents a potent and attractive tool for immunomonitoring, lending itself readily to automation and with a high throughput. This opens the possibility of an easy and reliable cytokine profiling for clinical applications.
Boeuf
Philippe
P
Unité d'Immunologie Moléculaire des Parasites, URA CNRS 2581, Département de Parasitologie et de Médecine Moléculaire, Institut Pasteur, 28 rue du Dr, Roux, 75724 Paris Cedex 15, France. pboeuf@pasteur.fr <pboeuf@pasteur.fr>
Vigan-Womas
Inès
I
Jublot
Delphine
D
Loizon
Séverine
S
Barale
Jean-Christophe
JC
Akanmori
Bartholomew Dicky
BD
Mercereau-Puijalon
Odile
O
Behr
Charlotte
C
D000328:Adult; D001331:Automation; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D002648:Child; D016207:Cytokines / Q000235:genetics*; D004247:DNA / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008263:Macrophage Migration-Inhibitory Factors / Q000235:genetics; D016778:Malaria, Falciparum / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D016212:Transforming Growth Factor beta / Q000096:biosynthesis / Q000235:genetics; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D016207:Cytokines; D008070:Lipopolysaccharides; D008263:Macrophage Migration-Inhibitory Factors; D012333:RNA, Messenger; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha; D004247:DNA