Rat pro-inflammatory cytokine and cytokine related mRNA quantification by real-time polymerase chain reaction using SYBR green

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.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.Cytokines are regulatory proteins, which play a key role in inflammatory responses either directly or by their ability to induce the synthesis of cellular adhesion molecules or other cytokines in numerous cell types. Knowledge of the local cytokine pattern is essential to elucidate the immune and pathological pathways involved in many inflammatory responses such as infectious diseases, autoimmune reactions, etc. However, cytokine protein detection, via techniques such as ELISA only allows the measurement of a limited number of cytokines from a single sample. In addition, tissue samples are often too small to enable their quantification at the protein level. Until now, this point has been a critical one. Processing of rat samples with ELISA techniques is also impaired by the lack of sensitivity of currently commercialized ELISA kits. Fortunately, the development of quantitative reverse transcription polymerase chain reactions (RT-PCR) provides a highly sensitive tool. Thus, quantification of mRNA is widely used to inve