Does UV cause DNA fragmentation? - how long do I need to put cells under UV to see fragmentation? (Feb/03/2009 )
nice to see you back here.
yes, I am pretty sure I see apoptosis by UV irradiation. I've checked with other methods.....I still think the kits give me genomic DNA instead of total DNA.
Genomic DNA is where you would expect to see the laddering, besides the ribosomal and mitochondial components are tiny compared to the genomic, so I would be surprised if you could see it in them.
if you take the dna fragmentation as a marker for apoptosis, you might want to keep in mind that UV light leads to double-strand-breakes which then in turn causes the intrinsic apoptotic pathway to kick in (if the damage is not repairable). Its not that the UV itself would induce strandbreaks, but it causes Thymine-dimers which can lead to failures during DNA repair and in the end, repair-enzyme failure leads to DSBs.
nevertheless, if apoptosis is triggered ( be it because of irradiation, chemicals or death-receptor ligands )it would lead to caspas3activity which also fragments the DNA.
if you want to visualize it ,you could try TUNEL-assay, it visualizes free DNA ends via fluorescence.
i used TNFalpha with success (20ng/ul for 6h in HEK293 cells)