Can anyone suggest me a simple yet efficient protocol for DNA fragmentation assay to confirm apoptotic induction in cancer cell lines, with reference?
You could try the protocol of Gooch JL, Yee D (Strain-specific differences in formation of apoptotic DNA ladders in MCF-7 breast cancer cells. Cancer Lett 144: 31–37, 1999). I tested it and I think is very simple and efficient protocol.
THIS IS VERY SIMPLE AND GOOD PROTOCOL
ESTIMATION OF DAMAGE IN DNA
The method proposed by Chang et al. (2002) was used to determine DNA strand breaks.
REAGENTS
1. DNA (2g)
2. Tris buffer (30mM, pH 7.4)
3. H2O2 (30%)
4. FeCl3 (500M)
5. Agarose (1%) in 1X TAE buffer
6. EtBr (10mg/ml)
7. Gel loading dye (0.25% bromophenol blue, 0.25% xylene cyanol, 50% glycerol)
8. 50X TAE buffer (Tris base 24.2g, EDTA 18.612g, glacial acetic acid 5.71ml in a total volume of 100ml, pH 8.0)
PROCEDURE
The reaction was carried out in tris buffer (pH 7.4) at 37C. Hydroxyl radicals were generated by allowing FeCl3 and H2O2 to react. The reaction mixture contained 5µl of tris buffer or DNA (2µg) and 5µl of tris buffer or leaf extracts followed by the addition of 10µl H2O2 and 5µl of FeCl3. DNA was then incubated at 37C for 30 minutes. To the above mixture, 6µl of loading dye was added and electrophoresed in 1% agarose gel containing 3µg/ml EtBr, at 100V for 15 minutes. DNA bands were viewed under transilluminating UV light and photographed using an Alpha Digidoc digital gel documentation system (Alpha Innotech, USA). The intensity of the bands were quantified using the software of the system.
DNA fragmentation by carcinogens even before apoptosis occurs can be seen in comet assay. it needs quite a lot preparation, but gives substantial knowledge on the level of dna fragmentation, worth it if you are going to use it in your next projects. See at: http://mutage.oxfordjournals.org/content/23/3/143.short
http://www.sciencedirect.com/science/article/pii/0165111094000133
Apoptose can be easily checked by PARP or Caspases activation.... Except if you just want to "see" the DNA fragmentation....
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