[Real-time fluorescent quantitative PCR for detection of peripheral blood T-cell receptor excision circles].
OBJECTIVE
To develop and optimize real-time fluorescence quantitative PCR (FQ-PCR) with the housekeeping gene RAG2 as cell number control to quantify T-cell receptor excision circle (TREC) in the peripheral blood.


METHODS
The real-time PCR system for amplifying TREC and RAG2 genes was established on the basis of ABI 7000 apparatus using Golden Taq system, designed primers, TaqMan-MGB probes and optimized buffer. PCR conditions were optimized with standard samples of TREC plasmid.


RESULTS
The amplification with the primer pair T(3) and T(4) was more efficient than that with T(1) and T(2). More specific and efficient amplification in FQ-PCR was achieved using TaqMan-MGB probes as compared with general Taq-Man probes. Golden Taq was more effective than general Taq in improving the specificity and decreasing the artifact, and 95 degrees C; for 10 min, 95 degrees C; for 5 s, and 53 degrees C; for 30 s for a total of 40 cycles using ABI7000 was found as the optimized thermal parameter setting.


CONCLUSIONS
An optimized real-time PCR protocol for detecting TREC in peripheral blood mononuclear cells is established.
Yin
Ji-xia
JX
Medical Laboratory Center, Nangfang Hospital, Southern Medical University, Guangzhou 510515, China.
Wu
Da-lin
DL
Xu
Wen-juan
WJ
Sun
Jing
J
D000293:Adolescent; D000328:Adult; D004268:DNA-Binding Proteins / Q000235:genetics*; D005260:Female; D015329:Gene Rearrangement, T-Lymphocyte / Q000235:genetics*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009687:Nuclear Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D015203:Reproducibility of Results
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C499311:RAG2 protein, human; D011948:Receptors, Antigen, T-Cell