[Real-time quantitative PCR for evaluating murine thymic function].
OBJECTIVE
To establish a real-time quantitative PCR method for detecting the levels of the signal joint T cell receptor excision circles (sjTRECs) in murine thymocytes and spleen lymphocytes for determining the amount of naive T cells and evaluating the thymic function.


METHODS
The genomic DNA was extracted from murine thymocytes and splenocytes for PCR amplification of the target fragments. After purification of the PCR product, the recombination-activating gene 2 (RAG(2)) fragment was cloned into pGEMT-Easy vector to construct the standard plasmid. After PCR optimization, the standard curve was obtained and the samples (thymocytes and splenocytes of BALB/c and C(57)BL/6 mice) were detected for sjTRECs by real-time quantitative PCR.


RESULTS
The standard plasmid was correctly constructed, and the standard curve with high reliability was obtained. No statistical difference was observed in sjTREC contents in the T lymphocytes between the two mouse strains.


CONCLUSIONS
Real-time quantitative PCR for sjTREC analysis is established successfully, which offers an important means for thymic function analysis and a reliable model establishment for study the thymus.
Institute of Tissue Transplantation and Immunology, Ji'nan University, Guangzhou 510632, China. huahuazhang@126.com
Hua-hua
HH
Zhang
Yao-yin
YY
Zeng
Xian-hui
XH
He
Fei-yue
FY
Xing
D000818:Animals; D015329:Gene Rearrangement, T-Lymphocyte; D018655:Lymphocyte Count; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction; D011948:Receptors, Antigen, T-Cell / Q000235:genetics* / Q000378:metabolism; D013601:T-Lymphocytes / Q000166:cytology*; D013950:Thymus Gland / Q000166:cytology* / Q000276:immunology
D011948:Receptors, Antigen, T-Cell