mRNA PCR-based epitope chase method

Methods Mol Biol. 2013:969:305-20. doi: 10.1007/978-1-62703-260-5_19.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • Animals
  • CD4-Positive T-Lymphocytes / cytology
  • CD4-Positive T-Lymphocytes / immunology*
  • CD8-Positive T-Lymphocytes / cytology
  • CD8-Positive T-Lymphocytes / immunology*
  • DNA, Complementary / chemistry
  • DNA, Complementary / genetics
  • DNA, Complementary / immunology
  • Electroporation / methods
  • Epitope Mapping / methods*
  • Epitopes, T-Lymphocyte* / genetics
  • Epitopes, T-Lymphocyte* / immunology
  • Humans
  • RNA, Messenger* / chemistry
  • RNA, Messenger* / genetics
  • RNA, Messenger* / immunology
  • Reverse Transcriptase Polymerase Chain Reaction / methods*

Substances

  • DNA, Complementary
  • Epitopes, T-Lymphocyte
  • RNA, Messenger