An accurate quantitative method for screening effective siRNA probes targeting a Hepatitis B virus transcript in single living cells.
A dual fluorescence reporter plasmid expressing EGFP and DsRed-Monomer from separate promoters was constructed for quantitative flow cytometry analysis. Cloning the hepatitis B virus (HBV) X gene into the 3' UTR region of DsRed-Monomer allowed quantifying the efficacy of ten siRNAs designed according to the accessibility of HBx mRNA measured in vitro. Using EGFP as an internal control, a justified calculation of the changed mean fluorescence intensity of DsRed-Monomer in each transfected cell yielded highly consistent results, and revealed all 10 siRNAs achieved over 50% inhibition among which a super effective siRNA achieved 88% inhibition at a very low concentration (0.33 microg/ml). This provides a quantification method critical for therapeutic application of siRNA.
Tong
Wen-Ping
WP
State Key Laboratory of Virology and Department of Biotechnology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, China.
Zhou
Yu
Y
Wang
Xinxin
X
Yang
Fan
F
Wu
Kai-Lang
KL
Wu
Jianguo
J
Zhang
Yi
Y
D005434:Flow Cytometry / Q000379:methods*; D018390:Gene Targeting / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics; D015347:RNA Probes / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics*; D014162:Transfection / Q000379:methods; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics*
D015347:RNA Probes; D034741:RNA, Small Interfering; D015534:Trans-Activators; D014157:Transcription Factors; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein