Negative control for Knockdown - (Feb/21/2007 )
There are 2 main types of negative controls for siRNA that I know of:
1) universal negative control that is not known to target any known gene
2) scrambled sequence of the target gene
Does anyone experience any non-specific effects of your negative control?
I have also heard about people who mutated 3 bases of the siRNA sequence and use it as a negative control, after they have verified that the mutant does not knockdown their target gene. Have anyone out there have experience with this? How to decide which bases to mutate?
I our lab, we use siRNA directed against another species's sequence as a negative control. (We do the knockdown in a mouse cell line, and use a siRNA directed against the human form of the protein as a control). Works fine.
we used luciferase shRNA as negative control.
Hi, I think It is not a good idea to mutated 3 bases and than use it as a negative control - do not forgot microRNA mechanism that works with not 100% complementarity and it is known that some siRNA are processed like miRNAs. This can bring problems. IT is better to use one of other mentionted possibilities.Marco
at least for mismatches are needed to avoid miRNA effect.
Scramble siRNA sequence is better.