Real-time fluorescent quantitative PCR for detection of peripheral blood T-cell receptor excision circles / 南方医科大学学报
Journal of Southern Medical University
;
(12): 1009-1013, 2006.
Article
in Chinese
| WPRIM
| ID: wpr-335006
ABSTRACT
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>An optimized real-time PCR protocol for detecting TREC in peripheral blood mononuclear cells is established.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Leukocytes, Mononuclear
/
Nuclear Proteins
/
Receptors, Antigen, T-Cell
/
Gene Rearrangement, T-Lymphocyte
/
Polymerase Chain Reaction
/
Reproducibility of Results
/
DNA-Binding Proteins
/
Genetics
/
Metabolism
/
Methods
Type of study:
Diagnostic study
Limits:
Adolescent
/
Adult
/
Female
/
Humans
/
Male
Language:
Chinese
Journal:
Journal of Southern Medical University
Year:
2006
Type:
Article
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