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mRNA PCR-based epitope chase method.

Authors
  • Doucet, Jean-Daniel
  • Gauchat, Dominique
  • Lapointe, Réjean
Type
Published Article
Journal
Methods in Molecular Biology
Publication Date
Jan 01, 2013
Volume
969
Pages
305–320
Identifiers
DOI: 10.1007/978-1-62703-260-5_19
PMID: 23296942
Source
Medline
License
Unknown

Abstract

The identification of specific viral and tumor antigen epitopes recognized by CD4(+) or CD8(+) T lymphocytes remains a challenge. Unfortunately, epitope mapping methods are generally costly and time-consuming. This chapter details a polymerase chain reaction (PCR)-based mRNA epitope identification method called mPEC, which is designed to rapidly and precisely identify relevant T cell epitopes recognized by previously isolated CD8(+) or CD4(+) T lymphocytes.This method is based on the use of mRNA fragments synthesized from PCR-amplified cDNA with a variety of 3'end iterative deletions. mRNA fragments are electroporated into autologous antigen-presenting cells to map the epitope in a given protein antigen. Considering mRNA's sensitivity to degradation, we also insert a control define epitope at the mRNA's 3'end to control for electroporated mRNA's integrity and capacity to be translated.

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