[An efficient method for screening effective siRNAs using dual-luciferase reporter assay system].
OBJECTIVE
To establish an efficient method for screening effective small interference RNA (siRNA) using dual-luciferase reporter assay system.


METHODS
Based 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.


RESULTS
In 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).


CONCLUSIONS
The dual-luciferase reporter assay system provides a useful method for screening effective siRNAs targeting specific genes.
Shan
Zhi-xin
ZX
Research Center of Medical Sciences, Guangdong Provincial People's Hospital/Guangdong Provincial Cardiovascular Diseases Institute, Guangzhou 510080, China. zhixinshan@yahoo.com.cn
Lin
Qiu-xiong
QX
Tan
Hong-hong
HH
Deng
Chun-yu
CY
Liu
Xiao-ying
XY
Xiao
Ding-zhang
DZ
Yu
Xi-yong
XY
D000818:Animals; D002460:Cell Line; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D008156:Luciferases / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection
D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D008156:Luciferases