Application of real-time quantitative PCR in selection of transfected cell strains for transgenic overexpression.
- Author:
Shao-Yan HU
1
;
Zi-Xing CHEN
;
Ye ZHAO
;
Wei-Ying GU
;
Jian-Nong CEN
;
Jun QIAN
Author Information
1. Jiangsu Institute of Hematology, The First Affiliated Hospital of Suzhou University, Suzhou 215006, China.
- Publication Type:Journal Article
- MeSH:
Blotting, Western;
Carrier Proteins;
genetics;
metabolism;
Gene Expression Regulation, Neoplastic;
Humans;
K562 Cells;
Nuclear Proteins;
genetics;
metabolism;
Organic Chemicals;
chemistry;
RNA, Neoplasm;
metabolism;
Retinoblastoma-Binding Protein 7;
Reverse Transcriptase Polymerase Chain Reaction;
methods;
Transfection;
Transgenes;
genetics
- From:
Journal of Experimental Hematology
2005;13(6):1062-1066
- CountryChina
- Language:Chinese
-
Abstract:
To explore the feasibility of real-time quantitative PCR (QRT-PCR) for selecting cell strains which overexpress a certain transgene, expression level of RbAp46 was detected in transfected cell strains by using optimal real-time PCR with SYBR Green I. Meanwhile, semi-quantitative RT-PCR and Western blot were performed to compare with the QRT-PCR. The results showed that values of RbAp46(N) were 2064.42 +/- 253.47, 860.94 +/- 291.07, 234.456 +/- 31.08, 18.17 +/- 5.14 and 1.46 +/- 0.54 in K562/RbAp46, K562/CMV, SHG44/RbAp46 monoclone, SHG44/RbAp46 multiclone and SHG44/CMV, respectively. The results were consistent with that determined by semi-quantitative RT-PCR and Western blot. It is concluded that QRT-PCR provides a highly efficient and reproducible method for selection of transfected cell subclones at different level of transgene expression.
