Real-time quantitative PCR for evaluating murine thymic function.
- Author:
Hua-hua ZHANG
1
;
Yao-yin ZENG
;
Xian-hui HE
;
Fei-yue XING
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Gene Rearrangement, T-Lymphocyte; Lymphocyte Count; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Polymerase Chain Reaction; Receptors, Antigen, T-Cell; genetics; metabolism; T-Lymphocytes; cytology; Thymus Gland; cytology; immunology
- From: Journal of Southern Medical University 2006;26(1):62-65
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo 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.
METHODSThe 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.
RESULTSThe 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.
CONCLUSIONSReal-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.
