Quantification of murine IFN-gamma mRNA and protein expression: Impact of real-time kinetic RT-PCR using SYBR Green I dye

Reliable quantification of cytokine mRNA expression is an important techniq ue for analyzing immune responses. Up until now, little to no information h as been available as to whether different mRNA quantification techniques le ad to similar results. Recently, real time quantitative reverse transcripta se (RT)-PCR using SYBR (R) Green I as a double stranded DNA specific dye ha s been introduced. This novel method enables simple and rapid measurement o f PCR product accumulation during the log-linear reaction phase and obviate s the need for expensive hybridization probes. Here, we analyzed murine gam ma interferon (IFN)-gamma mRNA expression in splenocytes by this technique in comparison to semiquantitative noncompetitive RT -PCR, Northern blot ana lysis, and ELISA after stimulation of the cells with interleukin (IL)-12, I L-18 and a combination of both cytokines. The results clearly show that all of the techniques detect differences in the IFN-gamma gene expression indu ced by these distinctive stimuli qualitatively exactly in the same order. H owever, real-time kinetic RT-PCR offers several advantages, notably its hig h sensitivity that allows the detection of basal IFN-gamma mRNA expression in unstimulated samples. In addition it provides the lowest interassay vari ability of all techniques investigated. Finally, the gene expression measur ed by this method eliminates any post-PCR manipulations because the PCR pro duct identification can be easily performed by melting curve analysis.