[Application of real-time quantitative PCR in selection of transfected cell strains for transgenic overexpression].
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.
Hu
Shao-Yan
SY
Jiangsu Institute of Hematology, The First Affiliated Hospital of Suzhou University, Suzhou 215006, China.
Chen
Zi-Xing
ZX
Zhao
Ye
Y
Gu
Wei-Ying
WY
Cen
Jian-Nong
JN
Qian
Jun
J
D052160:Benzothiazoles; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003959:Diamines; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020014:K562 Cells; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D009930:Organic Chemicals / Q000737:chemistry; D011804:Quinolines; D012334:RNA, Neoplasm / Q000378:metabolism; D056524:Retinoblastoma-Binding Protein 7; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014162:Transfection; D019076:Transgenes / Q000235:genetics
D052160:Benzothiazoles; D002352:Carrier Proteins; D003959:Diamines; D009687:Nuclear Proteins; D009930:Organic Chemicals; D011804:Quinolines; C490746:RBBP7 protein, human; D012334:RNA, Neoplasm; D056524:Retinoblastoma-Binding Protein 7; C098022:SYBR Green I