mRNA PCR-based epitope chase method

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 969; p. 305
• Main Authors: Doucet, Jean-Daniel, Gauchat, Dominique, Lapointe, Réjean
• Format: Journal Article
• Language: English
• Published: United States 2013
• Subjects: 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; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA, Messenger - chemistry; RNA, Messenger - genetics; RNA, Messenger - immunology
• ISSN: 1940-6029
• DOI: 10.1007/978-1-62703-260-5_19

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.