title: Simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) by real-time PCR.
abstract: T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/10(6) cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.
authors.affiliation: CREA, Diagnostics Department, Spedali Civili di Brescia.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Sottini
authors.affiliation: CREA, Diagnostics Department, Spedali Civili di Brescia.
authors.forename: Federico
authors.initials: F
authors.lastname: Serana
authors.affiliation: CREA, Diagnostics Department, Spedali Civili di Brescia.
authors.forename: Diego
authors.initials: D
authors.lastname: Bertoli
authors.affiliation: CREA, Diagnostics Department, Spedali Civili di Brescia.
authors.forename: Marco
authors.initials: M
authors.lastname: Chiarini
authors.affiliation: CREA, Diagnostics Department, Spedali Civili di Brescia.
authors.forename: Monica
authors.initials: M
authors.lastname: Valotti
authors.affiliation: CREA, Diagnostics Department, Spedali Civili di Brescia.
authors.forename: Marion
authors.initials: M
authors.lastname: Vaglio Tessitore
authors.affiliation: CREA, Diagnostics Department, Spedali Civili di Brescia; luisa.imberti@spedalicvili.brescia.it.
authors.forename: Luisa
authors.initials: L
authors.lastname: Imberti
mesh_terms: D000293:Adolescent; D000328:Adult; D001402:B-Lymphocytes / Q000276:immunology / Q000502:physiology; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D004270:DNA, Circular / Q000032:analysis* / Q000097:blood / Q000235:genetics; D005260:Female; D006801:Humans; D008875:Middle Aged; D008969:Molecular Sequence Data; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D011948:Receptors, Antigen, T-Cell / Q000097:blood / Q000235:genetics*; D011995:Recombination, Genetic; D013601:T-Lymphocytes / Q000276:immunology / Q000502:physiology; D055815:Young Adult
chemical_list: D004270:DNA, Circular; D011948:Receptors, Antigen, T-Cell