Abstract
The following method describes the identification of candidate immunogenic peptides through their ability to recall an immune T-cell activation from peripheral blood mononuclear cells (PBMCs) of individuals with defined HLA–peptide restrictions that have been previously exposed to the antigen. Isolated PBMCs are plated out at a concentration of 1×106 cells/ml in a 200 μl medium and incubated overnight to reduce cytokine gene expression due to cell manipulation. After starving, cells are either directly stimulated with individual peptides or not stimulated and incubated from 3 to 12 h according to experimental conditions. Quantitative real-time PCR (qrt-PCR) is performed on reverse-transcribed complementary DNA (cDNA) from total RNA that is isolated from peptide-cultured PBMCs. To perform high quality qrt-PCR, primers and probes are designed to span exon–intron junctions in order to prevent amplification of genomic DNA and to produce amplicons <150 base pairs (bp). Real-time monitoring of fluorescent emission from the cleavage of sequence specific probe by the nuclease activity of Taq polymerase (TaqMan method) defines threshold cycles during exponential phases of amplification. Standard curves of copy numbers of the gene of interest are normalized using as reference copy numbers of control genes.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Provenzano, M., Spagnoli, G.C. (2009). T Cell Epitope-Mapping by Cytokine Gene Expression Assay. In: De Libero, G. (eds) T Cell Protocols. Methods in Molecular Biology™, vol 514. Humana Press. https://doi.org/10.1007/978-1-60327-527-9_8
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DOI: https://doi.org/10.1007/978-1-60327-527-9_8
Publisher Name: Humana Press
Print ISBN: 978-1-58829-587-3
Online ISBN: 978-1-60327-527-9
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