Team:REC-CHENNAI/Demonstrate
Demonstrate
Our project is about the use of an alternate transfection reagent, a chimeric Cell Penetrating Peptide [CPP] as an alternative to the commercial liposomal reagents due to their potential drawbacks. The experimental design involves mixing of chimeric Cell Penetrating Peptide called BP100-(KH)9 with the silencing RNA which targets the surviving mRNA. We carried out the experiments in HeLa cell line. The unconjugated cargo contains the short hairpin like silencing RNA which is conjugated with a fluorophore and a quencher. We validated our system by the following means 1. The emission of fluorescence by the short hairpin like silencing RNA due to the spatial separation of the fluorophore and the quencher due to unwinding on account of binding to the target mRNA. We transfected the unconjugated cargo using CPP. Fluorescence was observed and the intensity of emitted fluorescence was measured. 2. Confirmation of the degradation of the Survivin mRNA by Reverse Transcriptase PCR.