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about RNAi

Posted by iglezer on 25 Jun 2008 at 02:45 GMT

I would like to know if the authors tested different shRNA sequences targeting Grx1, or just the one described in methods. Since "off-target" is a main concern in RNAi experiments (a subject addressed in several reviews) and the empty vector was the only control, it seems to me that the RNA interference experiments are not conclusive. In addition, a "rescue" experiment would be even more important in this regard. The fact that Grx2 does not change with the shRNA does not exclude the possibility that other proteins are affected or that shRNA have toxic effects by itself. I am posting this comment because other researchers may follow the trend of performing RNAi without proper controls, which would not fit with PLoS ONE aims to publish well-performed science.

RE: about RNAi

Viji replied to iglezer on 04 Aug 2008 at 10:02 GMT

In response to the concerns raised by Iglezer we wish to report that we have performed experiments using scrambled ShRNA for Grx1 in addition to empty vector and we do not find redox modification of VDAC in cells transfected with either scrambled ShRNA or empty vector (We are unable to upload the figure here) As regards to the "rescue experiment" with Grx1 overexpression, we have indeed performed these experiments using a different experimental paradigm. The problem of doing double transfection (ShRNA and over-expression of Grx1) is that ShRNA reduces Grx1 expression by approximately 50% but over-expression leads to 10-20-fold increase which would potentially be overwhelming. Therefore, we exposed cells to the excitatory amino acid, L-BOAA , leading to loss of mitochondrial membrane potential and this was prevented by over-expression of Grx1 clearly demonstrating the role of Grx1 in maintenance of mitochondrial membrane potential.


RE: RE: about RNAi

Viji replied to Viji on 10 Aug 2008 at 09:56 GMT

For the reply to Iglezer which we had posted on 5th august 2008, we were unable to upload the relevant figure due to technical difficulties. The figure can be accessed through this link