Need help evaluating apoptosis data - detection of degraded DNA in cytoplasmic fraction (Dec/13/2006 )

Hello,

I have been doing experiments that show when I treat cancer cells with a particular siRNA and see that cells stop dividing. Did several apoptosis assays (Roche Cell Death ELISA) and see at my 48 hour timepoint about a 5-7 fold increase in apoptosis and by 72 hours I have seen 20 fold, 30 fold and 70 fold increase in apoptosis in siRNA compared to transfections with nontargeting RNA.

My question is does this seem valid? I have seen other people report apoptosis as a 20-30% increase not such a huge fold increase!

I have done westerns and get very nice PARP cleavage and an increase in proapoptotic BAK.

If anyone has any insight I would appreciate it. Thanks

-nancyd-

You should be very happy, if it is reproducible. It's huge % of apoptosis.

-exploresci-

usually with apoptosis assays, the most important thing one has to show is that its not necrosis - which is what most reviewers will argue. So if u can convince them that its really apoptosis, then u dont have to worry.

And u r using siRNA, so u will have to show abscence of off target effects. else it will b difficult to sell it to the reviewers.

good luck !!!

-scolix-

[quote name='scolix' date='Dec 14 2006, 11:35 AM' post='81078']
usually with apoptosis assays, the most important thing one has to show is that its not necrosis - which is what most reviewers will argue. So if u can convince them that its really apoptosis, then u dont have to worry.

And u r using siRNA, so u will have to show abscence of off target effects. else it will b difficult to sell it to the reviewers.

Thanks. For off target effects do you mean that my RNAi is effecting something other than my protein of interest? We do not see the effect using nontargeting RNA. We also do not see the effect in a cell type that is deficient in the target that my blocked protein is proposed to affect.

So does having a beautiful Western of PARP cleavage convince of apoptosis?

And are you guys SURE 70 fold apoptosis is believable?

-nancyd-

QUOTE (nancyd @ Dec 14 2006, 12:01 AM)

we also work with Roche DNA-histone ELISA; try also shorter incubation time f.i. 24 h; your values are conceivable in general, but is increase of 20-30% by others performed with the same siRNA, or what do you mean? ;
as suggested other specific apoptosis assays may confirm your data such as your additional assays; I think you will also perform Roche LDH assay to show both necrosis and apoptosis

-The Bearer-

with off target effects, i mean like interferon response.

With what u have told so far, I am unsure if one could find such a high % apoptosis in 48hrs -72 hrs. If its 30-50% , it still acceptable. Thats y u need to look at literature to prove ur point that its apoptosis. Have u looked at caspase (i think 3 and or 7) levels.

-scolix-