An accurate quantitative method for screening effective siRNA probes targeting a Hepatitis B virus transcript in single living cells

Biochemical and Biophysical Research Communications
Wen-Ping TongYi Zhang

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.

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Citations

Feb 24, 2016·The Journal of Clinical Investigation·Peter A Revill, Stephen A Locarnini

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