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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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siRNA - augmentaion instead of knockdown
Posted by: Lena (IP Hidden, Unregistered user, )
Date: April 2, 2005 07:51AM
I have tried to knockdown Luciferase gene using luciferase reporter plasmid in HEK293 cells and Oligonucleotide Luciferase 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 - 5000 (arbitrary units), with non related siRNA in the FIV vector -8000 and with siRNA OligonucleotideLuciferase -13,000 The results repeated twice. I would like to understand why did i get augmentation of the signal instead of knockdown of the gene. What i did wrong? Thank you for your help.
Re: siRNA - augmentaion instead of knockdown
Posted by: 5'-ATCG (IP Hidden, Unregistered user, )
Date: April 2, 2005 12:46PM
One key here is that you need to make sure every cell taking the luciferase reporter "must" also taking in sufficient amount of your siRNA expression vector (assuming that the expression level of these 2 plasmid is same). Otherwise, you won't see any knockout/knockdown effect. The optimal molar ratio of 2 plasmids for a typical co-transfection is 10:1 or 20:1. Good luck.
Re: siRNA - augmentaion instead of knockdown
Posted by: Lindsay (IP Hidden, Unregistered user, )
Date: April 3, 2005 11:21AM
your ~80% transfection efficiency is referring to your siRNA construct (GFP) but not Luc reporter? so efficieny of your Luc reporter may be much lower. Total 13 ug of DNA for co-transfection is too much to my impression (unless for 100mm- or 150-mm dish)??
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