Rat pro-inflammatory cytokine and cytokine related mRNA quantification by real-time polymerase chain reaction using SYBR green.
BACKGROUND
Cytokine mRNA quantification is widely used to investigate cytokine profiles, particularly in small samples. Real-time polymerase chain reaction is currently the most reliable method of quantifying low-level transcripts such as cytokine and cytokine receptor mRNAs. This accurate technique allows the quantification of a larger pattern of cytokines than quantification at the protein level, which is limited to a smaller number of proteins.


RESULTS
Although fluorogenic probes are considered more sensitive than fluorescent dyes, we have developed SYBR Green real-time RT-PCR protocols to assay pro-inflammatory cytokines (IL1a, IL1b and IL6, TNFa), cytokine receptors (IL1-r1, IL1-r2, IL6-r, TNF-r2) and related molecules (IL1-RA, SOCS3) mRNA in rats. This method enables normalisation against several housekeeping genes (beta-actin, GAPDH, CypA, HPRT) dependent on the specific experimental treatments and tissues using either standard curve, or comparative CT quantification method. PCR efficiency and sensitivity allow the assessment of; i) basal mRNA levels in many tissues and even decreases in mRNA levels, ii) mRNA levels from very small samples.


CONCLUSIONS
Real-time RT-PCR is currently the best way to investigate cytokine networks. The investigations should be completed by the analysis of genes regulated by cytokines or involved in cytokine signalling, providing indirect information on cytokine protein expression.
Peinnequin
André
A
Département de Radiobiologie et de Radiopathologie, Centre de Recherches du Service de Santé des Armées Emile Pardé, BP 87, 38702 La Tronche, France. andrepeinnequin@crssa.net
Mouret
Catherine
C
Birot
Olivier
O
Alonso
Antonia
A
Mathieu
Jacques
J
Clarençon
Didier
D
Agay
Diane
D
Chancerelle
Yves
Y
Multon
Eric
E
D000818:Animals; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics*; D017931:DNA Primers; D005456:Fluorescent Dyes; D007249:Inflammation / Q000235:genetics / Q000276:immunology; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D018121:Receptors, Cytokine / Q000096:biosynthesis / Q000235:genetics; D013997:Time Factors
D016207:Cytokines; D017931:DNA Primers; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D018121:Receptors, Cytokine