i am working on an anti cancer drug application on human lungs cancer cell lines (A549). i would like to do preliminary qPCR assay before WB.
i am confused of selecting detection of the required genes. Though i have finalized the followings: (BAK, BAX, BID, TNF-a, STAT-3, FADD)
Any suggestions for corrections or further addition would be of great help.
thanks
Kish
Hello Kishoree,
the apoptotic program also consists of post translational modifications of pro and anti-apoptotic proteins (e.g. phosphorylation, cleavage, translocation, prot.-prot. interaction). So the markers to be interrogated by western blot are not necessarily the same as by qPCR.
For example BAX/BAK from your list may not change expression, but increase of a pro-apoptotic protein (BID/BIM) may lead to their release, dimerization and MOMP. (Which, by the way, will be hard to prove in standard whole cell lysates as well).
So gene/protein expression alone will not give you the best idea of what's going on.
In general, I would go for cleavage of caspase 3 and PARP in WB, to confirm apoptosis. Annexin V /PI staining as alternative/complementary method. Doing qPCR would be really going into detail why and how apoptosis is induced and may turn out to be quite tricky.
Hope that helps.
Hello
i agree with Thomas Mühlenberg ,
And you can determine the expression levels of Bcl-2 and Bax mRNAs samples and evaluate possible differences in Bcl-2 and Bax mRNA levels at particular stages of cancer .
article here will help you .
Article Expression of cell survival/death genes: Bcl-2 and Bax at th...
Good Luck
I also agree with the above comments, essentially its very difficult to determine apoptosis just by qPCR analysis of target genes as many of these under go post translational modification, cleavage of pro or phosphorylation. I would suggest using an FAC's based assay AnV /pi or mitochondrial depolarisation JC-1 dye, great for FAC'S. There are also a number of caspase cleavage assays available for high throughput, caspase-3 cleavage of substrate releases a fluorescent marker, excellent for 96 well assays. If you are really set on qPCR analysis you need to include a cross section of pro and anti apoptotic bel-2 family members. It's a very fine balance between the expression of these and their interaction. I looked at this in the neutrophil worth a read Cowburn AS 2011. I found a survival factor increased the expression of pro apoptotic factors, priming the cell for apoptosis once the survival signal had subsided.
For qPCR Bax and Bak wouldn't help much discovering apoptosis as their apoptotic activity should be determined by their ratio to Bcl2. its better to check Bax/Bcl2 ratio on protein level. better to check P53 mRNA level as it is wild type on A549 cells so it would lead you better.
Right. You can make a case that a cell is priming itself for apoptosis, but due to the many mechanisms by which it might actually be triggered, and the interactions of key apoptotic regulators with alternative death pathways, you can never say for certain how the cells are dying (assuming you are seeing cell death) without biochemical analysis.
As it is said in the above comments, it is very difficult to determine apoptosis just by qPCR, as many of the proteins involved under go post translational modification, phosphorylation or cleavage activation. I would suggest an FAC's based assay such as AnV /pi or mitochondrial depolarisation dye, great for FAC'S. Also it is possible caspase cleavage assays for FAC'S.
- Badges
- Science topic