Samtidig Kvantificering af T-celle receptor Resektion cirkler (TRECs) og K-Sletning Recombination Resektion cirkler (KRECs) af Real-time PCR
Summary
Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.
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/106 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.
Introduction
T-celle-receptor excision cirkler (TRECs) og K-sletning rekombination excision cirkler (KRECs) er små cirkulariserede DNA-elementer, der er udskåret i en andel på T- og B-celler, henholdsvis i en genomisk DNA-rekombination proces, der fører til dannelsen af en meget forskelligartet repertoire af T- og B-celle receptorer. De har ingen funktion, men fordi de er stabile og ikke kan replikeres, er de fortyndes efter hver celledeling, således vedvarende kun i ét af de to datterceller. Derfor kan antages niveauet i perifert blod som et estimat af thymus og knoglemarv output.
Mens TREC analysen er stort set brugt de sidste 15 år for at vurdere omfanget af thymus output, til 1 på KRec analysen, der oprindeligt blev udviklet måle B-celle proliferation og dens bidrag til B-celle homeostase i sundhed og sygdom, 2 er først for nylig foreslået som en markør for knogle marrow output. 3,4 Her beskriver vi den metode, vi har udviklet til samtidig kvantificering af både TRECs og KRECs. 4
Med denne kombinerede fremgangsmåde Variabiliteten er forbundet til DNA kvantificering af real-time PCR elimineret ved anvendelse af en unik standardkurve fremstillet ved fortynding af en triple-insert plasmid indeholdende fragmenter af TRECs, KRECs og T-celle-receptor alpha konstant (TCRAC) gen i en 1: 1: 1 ratio. Dette tillader en mere nøjagtig vurdering af TREC og KRec kopiantal. Desuden samtidig kvantificering af de to mål i den samme reaktion tillader reduktion reagens omkostninger.
Den foreslåede TREC / KRec analysen kan være nyttigt at måle omfanget af T- og B-celle neo-produktion hos børn eller voksne med svær kombineret immundefekt (SCID), 4 Almindelig variabel immundefekt, 5 autoimmune sygdomme, 6-8 og HIV-infektion . 9 kan desuden bruges tilovervåge immune bedring efter hæmatopoietisk stamcelletransplantation, 10 enzymerstatningsterapi, 11 og antiviral 9 eller immunmodulerende behandlinger. 6-8 Endelig fordi SCID patienter indregnes ved TREC assay trods de underliggende genetiske defekter, og agammaglobulinæmi patienter kan identificeres under anvendelse KRec kvantificering Den TREC / KRec analysen kan også anvendes til at detektere immundefekter hos nyfødte screeningsprogrammer. 12. I dette tilfælde skal testen udføres på DNA ekstraheret fra små pletter af blod blottet og tørres på filtrerpapir, skal være meget følsom og specifik for de pågældende sygdomme, samt yderst reproducerbar og omkostningseffektiv.
Indførelsen af KRec kvantificering i testen bør forbedre opførelser af nyfødte screening for immundefekter, som rutinemæssigt blevet udført i nogle dele af USA (WI, MA, CA) siden 2008, hvor Wisconsin blev den første til at introduktionene analyse af TRECs i sit postnatal screeningsprogram. 13
Protocol
Representative Results
Discussion
TREC and KREC quantification can be considered a good estimate of recent thymic and bone marrow output provided that some caveats are taken into account. Even though an absolute quantification method employing standard curve requires more reagents and more space on the real-time PCR reaction plate, it ensures highly accurate quantitative results because unknown sample quantities are interpolated from standard curves built upon known amounts of starting material. Moreover this method is better fitted to detect low amount …
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors have no acknowledgements.
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Histopaque-1077 | Sigma Aldrich SRL | 10771-500 ML | density gradient separation method |
| QIAamp DNA Blood Mini Kit (250) | QIAGEN | 51106 | DNA extraction |
| Unmodified DNA Oligonucleotides HPSF 0.01 mmol | Eurofins MWG Operon/Carlo Erba Reagents S.r.l | Resuspend the lyophilized product to 100 picmol/µl | |
| AmpliTaq DNA Polymerase: including 10x Buffer II and 25mM MgCl2 | Applied Biosystems/Life-Technologies | N8080156 | |
| GeneAmp dNTP Blend (100 mM) | Applied Biosystems/Life-Technologies | N8080261 | |
| TOPO TA Cloning Kit for Subcloning | Invitrogen/Life-Technologies | K4500-01 | |
| XL1-Blue Subcloning Grade Competent Cells | Stratagene | 200130 | |
| PureYield Plasmid Miniprep System | Promega | A1223 | |
| SpeI 500U | New England Biolabs | R0133S | |
| HindIII-HF 10,000 U | New England Biolabs | R3104S | |
| PureYield Plasmid Midiprep System | Promega | A2492 | |
| XhoI 5,000 U | New England Biolabs | R0146S | |
| TRIS Utrapure | Sigma Aldrich SRL | T1503 | |
| EDTA | Sigma Aldrich SRL | E5134 | |
| TE buffer (1 mM TRIS and 0.1 mM EDTA) | |||
| TaqMan Universal PCR Master Mix | Applied Biosystems/Life-Technologies | 4364338 | |
| Dual labeled probes HPLC 0.01 mmol | Eurofins MWG Operon/Carlo Erba Reagents S.r.l | Resuspend the lyophilized product to 100 picmol/µl | |
| NanoDrop 2000c spectrophotometer | ThermoFisher | ||
| Applied Biosystems 2720 Thermal Cycler | Applied Biosystems/Life-Technologies | 4359659 | |
| Fast 7500 Real-Time PCR system | Applied Biosystems/Life-Technologies | ||
| SDS Sequence Detection Software 1.4 | Applied Biosystems/Life-Technologies |
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