[The quantitative detection of NKG2D, perforin and granzyme B gene expressions in lymphocytes by SYBR green I real-time fluorescence quantitative PCR].
OBJECTIVE
To develop a SYBR Green I real-time fluorescence quantitative PCR for the detection of lymphocyte immune function.


METHODS
The primers of NKG2D, perforin, granzyme B and keeping-home gene GAPDH were designed and synthesized according to NCBI gene sequences, and the real-time fluorescence quantitative PCR was established. The gene expressions of NKG2D, perforin and granzyme B in lymphocytes from cancer patients and CIK induced from the cancer patients lymphocytes in vitro were quantified by real-time fluorescence quantitative PCR.


RESULTS
NKG2D, perforin and granzyme B mRNA could be specifically amplified and quantitatively detected by the SYBR Green I real-time fluorescence quantitative PCR according to agarose gel electrophoresis and melt curve analysis. The mRNA expression of granzyme B was reduced in lymphocytes from cancer patients, however the mRNA expressions of perforin and granzyme B were increased in CIK induced by cytokines and monoclone antibody compared with their lymphocytes(P<0.01).


CONCLUSIONS
The SYBR Green I real-time fluorescence quantitative PCR is a useful method for the quantitative detection of NKG2D, perforin and granzyme B mRNA to investigate cellular immune function.
Department of Pathology, Kunming General Hospital of Chengdu Command, Kunming 650032, China. wenxing-zhao@sohu.com
Wen-xing
WX
Zhao
Ju-lun
JL
Yang
Xia
X
Li
Jin
J
Ai
Tao
T
Li
Li
L
Wang
D052160:Benzothiazoles; D003959:Diamines; D005260:Female; D005453:Fluorescence; D053804:Granzymes / Q000235:genetics*; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism*; D008297:Male; D055655:NK Cell Lectin-Like Receptor Subfamily K / Q000235:genetics*; D009930:Organic Chemicals; D054353:Perforin / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines
D052160:Benzothiazoles; D003959:Diamines; C527085:KLRK1 protein, human; D055655:NK Cell Lectin-Like Receptor Subfamily K; D009930:Organic Chemicals; D011804:Quinolines; D054353:Perforin; C098022:SYBR Green I; D053804:Granzymes