Journal: Signal Transduction and Targeted Therapy
Article Title: Peptide-substituted oligonucleotide synthesis and non-toxic, passive cell delivery
Figure Lengend Snippet: ( a ) A stably transfected vector in HeLa cells expresses two luciferase genes. Firefly luciferase is expressed constitutively, whereas expression of the renilla luciferase gene is under the control of miR-15b as part of the RNA-induced silencing complex (RISC). In the presence of an anti-miR-15b oligonucleotide (TP ODN in this figure), the RISC dissociates from the renilla gene. This leads to an increase in renilla luciferase activity. Comparison of the activity of renilla luciferase to the unregulated firefly luciferase yields an indication of the activity of an oligonucleotide as an anti-miR-15b oligonucleotide. ( a ) Adapted from the Integrated DNA Technologies Inc. (Coralville, IA, USA) website. The stably transfected HeLa cell line and assay protocols were developed by miRagen Therapeutics, Inc. ( b ) Lipid transfection using DharmaFECT 1 (Dharmacon). ( c ) Passive transfection using TP ODNs. Positive and negative controls were purchased from Dharmacon: miRIDIAN Hairpin Inhibitor (IH-300587-07) and miRIDIAN Hairpin Inhibitor Negative Control #1 (IN-001005-01).
Article Snippet: Passive transfection as in a dual-luciferase assay A TP ODN stock solution in water (Hyclone Cell Molecular biology Grade, Invitrogen) was added to 375 μl reduced serum without antibiotic medium (2% FBS, 1× GlutaMAX, 1× non-essential amino acids and 1× sodium pyruvate) so as to obtain a final concentration of 1 μM TP ODN.
Techniques: Stable Transfection, Transfection, Plasmid Preparation, Luciferase, Expressing, Activity Assay, IA, Negative Control