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Help: Advices on extracting RNA from pellets?


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#1 samir_munir

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Posted 13 July 2009 - 07:51 AM

Hello all labrats,

I am having an issue on isolating RNA from cells cultured in a pellets. I pelleted like 250.000 Mesenchymal stem cells and the let them culture for 3 weeks.
Terminated them with trizol and stored them at -20 C in a 2 ml ependorf tube.

Now i am having this issue: the pellet will not homogenize fuly. I have tried to vortex the pellet heavily, and then tried with 1 hours digetion with collagenase. I have also tried to combine the mentioned two procerdures with using a mixer mill. http://www.retsch.co...ction-features/

but the pellet will simply not disolve.

Any suggestions?

samir

#2 mdfenko

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Posted 13 July 2009 - 10:54 AM

why would you want everything to solubilize? you have membranes and other cell debris in your pellet. the rna (and dna and cytosolic proteins) should be extracted by the trizol.
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#3 cellcounter

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Posted 13 July 2009 - 11:30 AM

Hello all labrats,

I am having an issue on isolating RNA from cells cultured in a pellets. I pelleted like 250.000 Mesenchymal stem cells and the let them culture for 3 weeks.
Terminated them with trizol and stored them at -20 C in a 2 ml ependorf tube.

Now i am having this issue: the pellet will not homogenize fuly. I have tried to vortex the pellet heavily, and then tried with 1 hours digetion with collagenase. I have also tried to combine the mentioned two procerdures with using a mixer mill. http://www.retsch.co...ction-features/

but the pellet will simply not disolve.

Any suggestions?

samir

You can use handheld homogenizer and try to homogenize the pellet. It may still not go completely into solution, but at least you will get maximum out of it.

Vortex does not work in Trizol added to cell pellet and immediately frozen; I have faced the same issue earlier. Next time, resuspend the fresh cell pellet in Trizol before freezing it down. It takes half a minute.

Best.

#4 samir_munir

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Posted 13 July 2009 - 10:20 PM

Hello all labrats,

I am having an issue on isolating RNA from cells cultured in a pellets. I pelleted like 250.000 Mesenchymal stem cells and the let them culture for 3 weeks.
Terminated them with trizol and stored them at -20 C in a 2 ml ependorf tube.

Now i am having this issue: the pellet will not homogenize fuly. I have tried to vortex the pellet heavily, and then tried with 1 hours digetion with collagenase. I have also tried to combine the mentioned two procerdures with using a mixer mill. http://www.retsch.co...ction-features/

but the pellet will simply not disolve.

Any suggestions?

samir

You can use handheld homogenizer and try to homogenize the pellet. It may still not go completely into solution, but at least you will get maximum out of it.

Vortex does not work in Trizol added to cell pellet and immediately frozen; I have faced the same issue earlier. Next time, resuspend the fresh cell pellet in Trizol before freezing it down. It takes half a minute.

Best.


ahh... i was not aware of that vortex will not work if the trizol-tissue had been frozen down. I think I will try a handheld homeginizer and see how that will work. Hopefully I will need to digest the sample with collagenase then.

thanks for the answers :-)

samir




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