The Use of Real-Time Quantitative PCR for the Analysis of Cytokine mRNA Levels
Over the last decade, real-time-quantitative PCR (RT-qPCR) analysis has become the method of choice not only for quantitative and accurate measurement of mRNA expression levels, but also for sensitive detection of rare or mutated DNA species in diagnostic research. RT-qPCR is based on the standard principles of PCR amplification in addition to the use of specific probes or intercalating fluorescence dyes. At the end of every cycle, the intercalating dye binds to all double-stranded DNA. There is a quantitative relationship between the amount of starting DNA and the amount of amplification product during the exponential phase. However, to obtain meaningful RT-qPCR data, the quality of the starting material (RNA, DNA) and the analysis method of choice are of crucial importance. In this chapter, we focus on the details of RNA isolation and cDNA synthesis methods, on the application of RT-qPCR for measurements of cytokine mRNA levels using Sybr-Green I as detection chemistry, and finally, we discuss the pros and contras of the absolute quantification versus relative quantification analysis. RT-qPCR is a powerful tool, but it should be “handled” with care.
Key wordsReal-time-quantitative PCR Absolute quantification Relative quantification Primer efficiency Housekeeping gene
- 5.Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., Speleman, F. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3: RESEARCH0034.Google Scholar
- 14.Forlenza, M., de Carvalho Dias, J.D., Vesely, T., Pokorova, D., Savelkoul, H.F., Wiegertjes, G.F. (2008) Transcription of signal-3 cytokines, IL-12 and IFN alpha beta, coincides with the timing of CD8 alpha beta up-regulation during viral infection of common carp (Cyprinus carpio L). Mol. Immunol. 45: 1531–1547.PubMedCrossRefGoogle Scholar