I would like to investigate the expression of some genes express in apoptotic cells by RT-qPCR in order to compare the results obtained by flow-cytometry (Annexin V).
I didn't find in literature genes that are commonly over-expressed in all cells during apoptosis.
I work with primary testicular cells.
Have someone an idea of which genes could be good to analyse?
Thank you.
Don't discount the regulation of apoptosis that occurs at the protein level. I would suggest looking for activated (cleaved) effector caspases 3 and 7, activated intiator caspases 2, 8 and 9, or the status of targets upstream of them - APAF, Bax, Bak, Bid, Parp, etc. Depending on if you are looking at ER stress, mitochrondrial damage, FAS signaling, or inflammation, different initiating caspases will be activated.
If your experimental conditions are sublethal to the cells, you may detect changes in apoptotic gene expression. This type of chronic conditioning may make the cells more susceptible to apoptotic signaling by upregulation of pro-apoptotic genes or downregulation of anti-apoptotic genes. However, if the conditions are very stressful or harsh, the cells may die from acute apoptotis signaling before you see a change in gene expression. In this case, looking at proteins will be more useful.
Good luck!
One idea is analysis Tp53.It is a common gene involved in apoptosis and easy to do by PCR. Another idea is the expression of proteins related to apoptosis by immunohistoquemistry, some example are p53 and ING2.
Don't discount the regulation of apoptosis that occurs at the protein level. I would suggest looking for activated (cleaved) effector caspases 3 and 7, activated intiator caspases 2, 8 and 9, or the status of targets upstream of them - APAF, Bax, Bak, Bid, Parp, etc. Depending on if you are looking at ER stress, mitochrondrial damage, FAS signaling, or inflammation, different initiating caspases will be activated.
If your experimental conditions are sublethal to the cells, you may detect changes in apoptotic gene expression. This type of chronic conditioning may make the cells more susceptible to apoptotic signaling by upregulation of pro-apoptotic genes or downregulation of anti-apoptotic genes. However, if the conditions are very stressful or harsh, the cells may die from acute apoptotis signaling before you see a change in gene expression. In this case, looking at proteins will be more useful.
Good luck!
Activated (cleaved) caspases 3 and 7 or cleaved PARP. Any of the pro-apoptotic Bcl2 family members (Bax, Bad, Bim, PUMA, etc). If you want to look at this group, it wouldn't hurt to check and see if the anti-apoptotic Bcl2 family members (Bcl-2, Bcl-XL, etc) have decreased expression or if their levels remain the same.
Proapoptotic Bad Bax Bid Puma Noxa Smac/ Diablo, cytochrome c, or antiapoptotic Bcl 2, survivin.
Cytochrome c will be wise choice.
Try to calculate the ratio of expression of pro-apoptotic genes (like bax, bak) to anti-apoptotic genes (like bcl-2, bcl-xl, Mcl-1) using quantitative PCR.
Article Rapid detection of apoptosis through real-time reverse trans...
Activated/cleaved caspases 3, caspases 7, cleaved PARP, bax, bak, bid, puma noxa, bcl-2, bcl-xl, mcl-1, p53, and cytochrome c.
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