An efficient method for screening effective siRNAs using dual-luciferase reporter assay system.
- Author:
Zhi-xin SHAN
1
;
Qiu-xiong LIN
;
Hong-hong TAN
;
Chun-yu DENG
;
Xiao-ying LIU
;
Ding-zhang XIAO
;
Xi-yong YU
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Cell Line; Genes, Reporter; Green Fluorescent Proteins; genetics; Luciferases; genetics; Plasmids; genetics; RNA Interference; RNA, Small Interfering; genetics; Transfection
- From: Journal of Southern Medical University 2009;29(8):1577-1581
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo establish an efficient method for screening effective small interference RNA (siRNA) using dual-luciferase reporter assay system.
METHODSBased on the siRNA expression vector pSilencer-4.1, 3 candidate green fluorescence protein (GFP) gene siRNA expression plasmids, namely pSi-GFPsiRNA1, pSi-GFPsiRNA2, and pSi-GFPsiRNA3, along with the negative control pSi-Negative, were constructed. Using the pGL3-promoter vector, the GFP-luciferase (GFP-LUC) expression plasmid pGL3-GFPf was constructed with the same Kozak consensus translation initiation site and start codon ATG for GFP-LUC coding sequence. The GFP fragment containing the target sequences of 3 GFP siRNAs was introduced into the 3' untranslate region of LUC in the modified pGL3-promoter vector to construct the plasmid pGL3-GFPp. The GFP siRNAs expression plasmids and Renilla luciferase reporter vector pRL-TK were co-transfected with pGL3-GFPf or pGL3-GFPp into the HEK293 cells, respectively. The luciferase activities were determined by dual-luciferase reporter assay, and the GFP mRNA expressions were detected by real-time quantitative PCR.
RESULTSIn the groups cotransfected with GFP siRNAs expression plasmids and pGL3-GFPf, the luciferase activities were reduced obviously, and the reduction was more significant in cells transfected with GFPsiRNA1 compared with the control cells (P<0.01).GFP mRNA levels were also markedly lowered in cells transfected with GFPsiRNA1 as shown by real-time PCR (P<0.01). In addition, the results of dual-luciferase reporter assay and real-time PCR showed that among the groups cotransfected with GFP siRNAs expression plasmids and pGL3-GFPp, the GFP expression was inhibited most obviously by GFPsiRNA1 (P<0.01).
CONCLUSIONThe dual-luciferase reporter assay system provides a useful method for screening effective siRNAs targeting specific genes.