I want to perform a DNA fragmentation apoptosis assay from whole organism DNA prep of Aedes aegypti mosquito. Has anyone performed a similar procedure before even in nematodes or larvae of other organisms? I would like to get a general procedure and things to avoid.
I have never used mosquito as my model but you can try.
1. you need a large number of bugs, but I dont know if you can conclude anything with the results??
2. Follow my papers--they are doable, quantitative and reproducible.
Download any of these papers and you will find the method......
Mol. Cell. biochem, 2006; Archives of toxicology 2000; JPET 1996; Free Radical biology & Med, 2001 (Ray SD et al or Hickey E. et al)
Good luck!!!
I know that in plant cells, we perform TUNEL test. You can perform a homemade test, but it will be easier to buy Life biotechnologies Tunel quit, of course it costs a lot more
there are different methods for DNA fragmentation including comet assay, and agarose gel electrophoresis, I perform DNA fragmentation of mouse liver, cancer cell line.
you can find the procedure from the attached file
Hi
Below is the link for extraction of DNA from Aedes sps.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3658367/
DNA sample were analyzed by agarose gel electrophoresis and the bands were visualized by ethidium bromide staining to visualize fragmentation.
I would suggest TUNEL as well, and I would also do Annexin V-PI analysis (it wouldn't take long and is a fairly easy technique; there are easy-to-do kits available). All the best.
The DNA fragmentation assay is not quantitative for apoptosis. Also, this assay depends on the presence of active caspases in the insect cells. Are they present? I ask, because there is caspase independent death reported in some systems. SO, the kits suggested in above answers, are quantitative, but we found many false positives with TUNEL assay. The method you choose will also depend on the question you are trying to answer. Hope this helps
There are defferent methods for DNA fragmentation assay. You can use DNA ladder kit (is a fairly easy test) or the procedure from the attached file. I would suggest you Annexin V/PI staining assay because this is quntitatively test.
I read all of the above comments and thought this is the right time to clarify some issues. DNA fragmentation assay was first developed by Wylie et al. 1980 and then we developed our method around 1989 for in vivo use. Please visit Ray SD et. al., Tox. Appl. Pharm, 1990; Shen W et al, 1991 and 1992; Ray SD et al., JPET, 1996). Seems like there is a lot of confusion regarding this assay based on above comments.
One has to recognize that: DNA frag assay can be qualitative and quantitative. The assay that I suggested is quantitative; agarose gel is qualitative and all the assays are loaded with flaws (it doesn't matter which one it is). THE GOLD STANDARD IS HISTOPATHOLOGY AND ELECTRON MICROSCOPY. I do not believe in any other assay besides these two.
Agreed that millions have published papers using TUNNEL assay and other assays and have gotten funded but all assays have their own drawbacks. The main reason that everyone agree to disagree that apoptosis and necrosis always co-exist and these population of cells survive in the presence of other contaminating cells; truth is that to date decent techniques are unavailable to separate normal cells from [autophagic; apoptotic, pro-apoptotic, necrotic and pro-necrotic] cells. All assays are done with wild guess and the degree of bias in most publications is very high.
Now the question is what Nanda Aung wants to do? If he wishes to see some micro changes in a distinct mosquito organ (obviously mosquito model), then histopath is the best way to go. Although EM is superior to histopath. I believe grinding a bunch of mosquitoes and getting some data could be scientifically meaningless. If Mr. Nanda can specify what exactly is his goal, specific suggestions can be provided.
Hi,
Thanks everyone for the suggestions. In short, I am making Aedes aegypti mosquito UV sensitive. I did not plan to do apoptosis assay initially. I am using an enzyme to qualitatively measure UV induced damage in the mosquito larvae's DNA. However, I found that the UV sensitive mosquito larvae contained fragmented DNA. So I am wondering if apoptosis occurred. That's why I wanted to try an apoptotic assay. I am planning to run the DNA on agarose gel. I really don't need to do a quantitative measurement. Currently, I don't have a DNA ladder kit and I am short on budget. So all i really want to know is if I can do a simple DNA prep and run the DNA on the gel to qualitatively measure DNA fragmentation.
Yes--you can. Couple of things you can try!!!
1. Run gel with diff concentrations of agarose (1.2, 1.4, 1.6%)
2. Load DNA (5, 10, 15, 20 micrograms per lane)
3. You may need tons of larvae samples to to do all of these; if larvae samples are not a problem, do #1 & 2 above one by one.
4. DNA extraction can be done as long as you have buff. saturated phenol, chloroform, isoamyl alcohol, ethanol, few buffers.......you do not need to buy the expensive kit. When we did DNA frag assays in late 80's none of the kits were available.
5. There could be several reasons for Fragmented DNA:
(i) Baseline endonuclease may be high (CAD-caspase-activated DNAse)
(ii) Check out Calcium levels in your buffers
(iii) Make sure to include Na-EDTA in all buffers etc.
(iv) Minimize mechanical disruptions
(v) Try to do all manipulations in ice-cold temp
very best Luck! Lets know how your gel turned out??
Run dna on gel... You should see ladder ;) but to prove that this is apoptosis you should confirm it using another test. Ask someone for annexin v and pi or 7aad
If you have cells in suspension and access to a cytometer, you can stain the samples with propidium iodide and quickly see DNA fragmentation. In addition, you can perform the comet assay. However, I agree that run gel in agarose is the most simple that you can do... considering that all tecniques have some limitations.
You can measured DNA-fragmentation with the Nicoletti method using flow cytometry. Nicoletti et al. Journal of Immunological Methods, 139(1991) 271-279.
Sometime using terminal transferase you can obtain false positive. I think that it's better if you are sure that the caspase in present, and you decide, if TUNEL assay it's correct or not.
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