Journal: Molecular Therapy. Nucleic Acids

Article Title: A dual fluorescence-based reporter assay for real-time determination of siRNA- and antisense oligonucleotide-mediated knockdown

doi: 10.1016/j.omtn.2025.102631

Figure Lengend Snippet: FluoroDetect assay—Mechanism of action (A) Schematic workflow of the FluoroDetect assay. Fluorescence signal of mCherry reporter gene can be downregulated by siRNA while the green fluorescence signal of the EGFP remains stable. (B) DNA and mRNA sequence of the target motif used in the FluoroDetect C-rich (HSat3) assay containing the flanking enzymatic cutting sites (pink) and siRNA-binding sites (blue). Additionally, the siRNA and ASO sequences are shown (m: 2′ MeO modification, ∗: thiophosphate modification). (C) Proof of concept using HeLa cells. Fluorescence microscope images showing green and red fluorescence signal in samples treated with targeting siRNA (siHSat3), untargeted siRNA control pool (siCo), and an untreated control. Images were taken 48 h after transfection.

Article Snippet: A transfer plasmid coding only EGFP was cloned by inserting an EGFP sequence into the pCDH-3xFLAG-TERT (Addgene plasmid #51631, RRID: Addgene 51631) generously provided by Steven Artandi.

Techniques: Fluorescence, Sequencing, Binding Assay, Modification, Microscopy, Control, Transfection