12 replies to this topic

[vitalgene]

I was wondering, whether apoptosis can be proven by RT-PCR or qPCR, couldn't find good references, i understand inexpensive tools are available to detect it at protein levels.

[Curtis]

Depends. Some people might say yes, but I say no.

In my research apoptosis occurs within 6 hours, and we tried many famous intracellular pathway gene mRNAs but they didn't show any significant difference at that period of time. So we realized apoptosis was so fast that mRNA level wasn't affected much. So we did Real-Time PCR. We noticed, in our case, protein modification and translocation of intracellular proteins from cytoplasm to mitochondria caused apoptosis.

But we did observe DNA fragmentation. We also did AO-PI experiments.  

p53 is a famous gene, you can try that one with qPCR though, we never did.

[vitalgene]

Curtis, on Jun 25 2010, 05:09 PM, said:

Depends. Some people might say yes, but I say no.

In my research apoptosis occurs within 6 hours, and we tried many famous intracellular pathway gene mRNAs but they didn't show any significant difference at that period of time. So we realized apoptosis was so fast that mRNA level wasn't affected much. So we did Real-Time PCR. We noticed, in our case, protein modification and translocation of intracellular proteins from cytoplasm to mitochondria caused apoptosis.

But we did observe DNA fragmentation. We also did AO-PI experiments.  

p53 is a famous gene, you can try that one with qPCR though, we never did.

Thank you Curtis
it will surly save my time
take care
vg

[snoopyx]

Curtis, on Jun 25 2010, 10:09 AM, said:

...
So we did Real-Time PCR. We noticed, in our case, protein modification and translocation of intracellular proteins from cytoplasm to mitochondria caused apoptosis.
...

Hi Curtis may you explain this a bit more. I don't get the point. How can you show translocation of proteins via Real-Time PCR?

Thx

[Curtis]

oh sorry...I must've put that sentence in a wrong place during edit. we did not do Real-Time for translocation analysis of course. I did cellular fractionation to find that out. What I meant is that even real-time PCR didn't show any significant increase or decrease in expression levels.

snoopyx, on Jul 1 2010, 02:12 AM, said:

Hi Curtis may you explain this a bit more. I don't get the point. How can you show translocation of proteins via Real-Time PCR?

Thx

[pmaj]

Hi Vitalgene,

I also study apoptosis. I have done Real-Time PCR on genes that have been shown to be involved in apoptosis, such as p53, BAX, Fas, PUMA etc. to prove conclusively, you can also do the TUNEL assay, or some other apoptosis detecting kits that are available int he market which are essentially ELISAs (I have used one from ROCHE). Also you can observe the cellular morphology for distinct apoptotic patterns, such as membrane blebbing. Further more if you have access to a fluorecent microscope or a confocal microscope, you can look for condensed chromatin after labeling it.

Hope this helps

Pmaj

[snoopyx]

Curtis, on Jul 1 2010, 06:22 AM, said:

oh sorry...I must've put that sentence in a wrong place during edit. we did not do Real-Time for translocation analysis of course. I did cellular fractionation to find that out. What I meant is that even real-time PCR didn't show any significant increase or decrease in expression levels.

snoopyx, on Jul 1 2010, 02:12 AM, said:

Hi Curtis may you explain this a bit more. I don't get the point. How can you show translocation of proteins via Real-Time PCR?

Thx

I'll probably have to do some fractionation soon as well. Can I pm you if I have any question?
Thx

[Curtis]

sure, why not? I can give you my protocol.

my boss asked me to not use Dounce Homogenizer, so we used digitonin buffers and modified somebody's protocol for better results.

Dounce is very popular, but my boss doesn't like it. digitonin method is so easy

[snoopyx]

Curtis, on Jul 2 2010, 04:25 AM, said:

sure, why not? I can give you my protocol.

my boss asked me to not use Dounce Homogenizer, so we used digitonin buffers and modified somebody's protocol for better results.

Dounce is very popular, but my boss doesn't like it. digitonin method is so easy

I would like to avoid the dounce homogenizer as I work with infected cells and aerosol production should be avoided as much as possible. I tried some protocols with various centrifugation steps, but the results weren't to good. So if you have a working protocol it would be great if you can forward it to me. It's nothing urgent, the project should start in the fall....

[Curtis]

Subcellular Fractionation
Subcellular fractionation was performed as previously described
[40] , but with minor modifications. Infected cells were
collected at different time points and resuspended in mitochondrial
buffer (70 m M Tris-HCl, 0.25 M sucrose and 1 m M EDTA,
pH 7.4). An equal volume of ice-cold digitonin lysis buffer (2 mg/
ml, 19.8 m M EDTA, 0.25 M D -mannitol and 19.8 m M MOPS, pH
7.4) was added for 90 s. Samples were then centrifuged twice at
300 g for 5 min to pellet the nuclei. The supernatant was further
centrifuged at 17,000 g for 20 min to separate mitochondria from
the cytosol.

then add RIPA to your nuclei and mitochondria fractions

[snoopyx]

Curtis, on Jul 3 2010, 12:03 AM, said:

Subcellular Fractionation
Subcellular fractionation was performed as previously described
[40] , but with minor modifications. Infected cells were
collected at different time points and resuspended in mitochondrial
buffer (70 m M Tris-HCl, 0.25 M sucrose and 1 m M EDTA,
pH 7.4). An equal volume of ice-cold digitonin lysis buffer (2 mg/
ml, 19.8 m M EDTA, 0.25 M D -mannitol and 19.8 m M MOPS, pH
7.4) was added for 90 s. Samples were then centrifuged twice at
300 g for 5 min to pellet the nuclei. The supernatant was further
centrifuged at 17,000 g for 20 min to separate mitochondria from
the cytosol.

then add RIPA to your nuclei and mitochondria fractions

Thanks! That was quick. You didn't use any kind of protease inhibitor? I usually add the Mini Complete inhibitor cocktail from Roche. It probably won't disturb the protocol if I add it?

[Curtis]

I added Roche PI Tablets to digitonin buffer only.

[snoopyx]

Curtis, on Jul 5 2010, 02:29 AM, said:

I added Roche PI Tablets to digitonin buffer only.

Thx a lot Curtis for your help.