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T cell epitope-mapping by cytokine gene expression assay


Provenzano, M; Spagnoli, G C (2009). T cell epitope-mapping by cytokine gene expression assay. In: De Libero, G; Walker, J M. T cell protocols (2nd ed.). New York, US: Springer, 107-118.

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 x 10(6) cells/ml in a 200 microl 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.

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 x 10(6) cells/ml in a 200 microl 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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Additional indexing

Item Type:Book Section, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Urological Clinic
04 Faculty of Medicine > University Hospital Zurich > Division of Surgical Research
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:2009
Deposited On:07 Apr 2009 14:35
Last Modified:26 May 2017 15:32
Publisher:Springer
Series Name:Methods in Molecular Biology
Number:514
ISSN:1064-3745
ISBN:978-1-58829-587-3 (P) 978-1-60327-527-9 (E)
Publisher DOI:https://doi.org/10.1007/978-1-60327-527-9_8
Related URLs:http://opac.nebis.ch/F/?local_base=NEBIS&con_lng=GER&func=find-b&find_code=SYS&request=005844273
PubMed ID:19048216

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