Quantification of T-cell receptor excision circle DNA using fluorescence resonance energy transfer and the LightCycler system.
T-cell receptor excision circles (TRECs) are circular, stable extrachromosomal DNA fragments and are generated during V(D)J gene recombination, a process responsible for diversity of the T-cell receptor repertoire. Here we describe a sensitive, rapid and easy to perform real-time PCR assay based on the LightCycler technique for the quantification of TRECs among peripheral blood cells and a comparison of this assay with conventional PCR-ELISA. By LightCycler, a sensitivity of 20 copies of plasmid deltaRec-psiJalpha Signal Joint TREC DNA was achieved whereas by PCR-ELISA, a detection limit of 2 copies was demonstrated. In blood samples from healthy individuals (n=52) a median TREC count of 1.6x10(4) copies [range 2x10(1) to 2x10(5)]/2x10(5) peripheral blood mononuclear cells (PBMNCs) and in cord blood, a median TREC count of 1.45x10(5) copies [range 1.2x10(5) to 1.6x10(5)]/2x10(5) PBMNCs) could be detected. No significant difference was found in 15 individuals when unfractionated PBMNCs (median count of 1.1x10(5) copies/2x10(5) PBMNCs) or magnetic associated cell sorter (MACS)-sorted CD45RA+ T-cells (median count of 2.5x10(5) copies/2x10(5) cells) were analyzed for TREC counts. In addition, we examined the number of deltaRec-psiJalpha Signal Joint TREC in paediatric (n=6) and adult patients (n=7) after allogeneic stem cell transplantation. In children, we observed a median TREC count of 5.7x10(4) copies/2x10(5) PBMNCs after 1x10(4) and 5.6x10(4) copies/2x10(5) PBMNCs after 2 years, and in adults, a median count of 3.6x10(4) copies/2x10(5) PBMNCs after 1x10(4) and 1.1x10(4) copies/2x10(5) PBMNCs after 2 years. In conclusion, the LightCycler-based real-time PCR assay described offers a very sensitive and rapid tool for the quantification of TREC DNA.
Loeffler
Juergen
J
Medizinische Klinik, Abteilung II, Otfried-Mueller-Str. 10, Eberhard-Karls-Universität, 72076 Tübingen, Germany. juergen.loeffler@med.uni-tuebingen.de
Bauer
Ralf
R
Hebart
Holger
H
Douek
Daniel C
DC
Rauser
Georg
G
Bader
Peter
P
Einsele
Hermann
H
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002648:Child; D002675:Child, Preschool; D004270:DNA, Circular / Q000097:blood* / Q000235:genetics / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005312:Fetal Blood / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D016867:Immunocompromised Host; D007223:Infant; D007231:Infant, Newborn; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods; D011948:Receptors, Antigen, T-Cell / Q000097:blood / Q000235:genetics* / Q000276:immunology; D033581:Stem Cell Transplantation / Q000009:adverse effects; D013950:Thymus Gland / Q000276:immunology
D004270:DNA, Circular; D011948:Receptors, Antigen, T-Cell