Quantitative polymerase chain reaction (qPCR) is an important technique for analysing target gene expressions in comparison to housekeeping genes. The aims of this project are to use this technique in order to investigate the expression of novel immunomodulatory mediators in isolated human immune cells (e.g. peripheral blood mononuclear cells, basophils etc.). Since individual cell type-specific mRNA expressions require homogeneous cell populations, a further aim of this project is to learn how to isolate and purify human immune cells, particularly basophils. Cells will be obtained from buffy coat blood purchased from the Deutsches Rotes Kreuz e.V. Leukocytes will be isolated by Ficoll-density centrifugation and certain cell types (e.g. basophils) further purified by immunomagentic cell sorting. Cells will then by lysed followed by isolation of total RNA and subsequent synthesis of cDNA, after which qPCR analysis will be performed. The project is expected to give students a comprehensive insight into analysing mRNA expressions, immune cell isolation and basic skills in literature reviews and data analysis.
Lit. Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods. 2001 Dec;25(4):386-401. Review.
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002 Jun 18;3(7):RESEARCH0034. Epub 2002 Jun 18.
The course is part of admission "Anmeldung gesperrt (global)".
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