title: An accurate quantitative method for screening effective siRNA probes targeting a Hepatitis B virus transcript in single living cells.
abstract: 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.
authors.affiliation: State Key Laboratory of Virology and Department of Biotechnology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, China.
authors.forename: Wen-Ping
authors.initials: WP
authors.lastname: Tong
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhou
authors.forename: Xinxin
authors.initials: X
authors.lastname: Wang
authors.forename: Fan
authors.initials: F
authors.lastname: Yang
authors.forename: Kai-Lang
authors.initials: KL
authors.lastname: Wu
authors.forename: Jianguo
authors.initials: J
authors.lastname: Wu
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
mesh_terms: 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*
chemical_list: 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