Protocol Online logo
Top : Forum Archives: : siRNA, microRNA and RNAi

siRNA - augmentation instead of knockdown - siRNA - augmentation instead of knockdown (Apr/02/2005 )

I have tried to do knockdown to Luciferase gene using luciferase reporter plasmid in HEK293 cells and OligonucleotideLuciferase provide to me in the kit "pFIV-H1-copGFPT siRNA Cloning and Expression Vector".
I used 3ug of luciferase reporter plasmid and 10 ug of the siRNA using CaCl2 transfection.
After 48 hours the cells were observed under the fluorescent microscope (transfection effeciency was >80%), but when i monitored luciferase activity the measurements without siRNA were - 6000 (arbitrary units), with non related siRNA in the FIV vector -8000 and with siRNA OligonucleotideLuciferase -12,000
The results repeated twice.

I would like to understand why did i get augmentation of the signal instead of knockdown of the gene.
What was wrong?



Where does your siRNA target to? Have you checked there is any homology, even partial, to other genes and non-coding regions?


Molar ratio of Luc reporter to siRNA vector is a key. Make sure each cell getting Luc also getting sufficient siRNA......