[Real-time fluorescent quantitative PCR for detection of peripheral blood T-cell receptor excision circles]
Ji-xia Yin1, Da-lin Wu, Wen-juan Xu
1Medical Laboratory Center, Nangfang Hospital, Southern Medical University, Guangzhou 510515, China.
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.
Conclusion:
An optimized real-time PCR protocol for detecting TREC in peripheral blood mononuclear cells is established.
