title: mRNA PCR-based epitope chase method.
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.
authors.affiliation: Research Centre, Centre Hospitalier de l'Université de Montréal (CRCHUM), Hôpital Notre-Dame, Université de Montréal and Institut du Cancer de Montréal (ICM), Montreal, QC, Canada.
authors.forename: Jean-Daniel
authors.initials: JD
authors.lastname: Doucet
authors.forename: Dominique
authors.initials: D
authors.lastname: Gauchat
authors.forename: Réjean
authors.initials: R
authors.lastname: Lapointe
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D018274:Electroporation / Q000379:methods; D018604:Epitope Mapping / Q000379:methods*; D018984:Epitopes, T-Lymphocyte / Q000235:genetics / Q000276:immunology; D006801:Humans; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D018076:DNA, Complementary; D018984:Epitopes, T-Lymphocyte; D012333:RNA, Messenger