Hi,
I`m trying to isolate RNA from spleen and thymus tissue.I already read that there is a problem with RNA degradation
due to high amount of RNAse in these tissues. And Yes, that`s also my problem.
I used the Trizol method.
Cutted a piece of the frozen tissue (around 60mg) and homogenized it in 1ml Trizol reagens.
Result: good RNA amount, 260/280..., no DNA left but also RNA broken down.
What can i do to prevent this degradation?
Use even less tissue?
I also found a tip to keep the Trizol cool might help.
Do you have any tips?
Thanks
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2 replies to this topic
#2
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Posted 17 December 2010 - 05:32 AM
Should not be this problematic. Maybe the tissue you started with were already degraded. Where the tissues were kept, were they in RNAlater?
Try LyseNow RNA kit or RNASound additives from Metammune, you can homogenize the tissue upfront and keep ltsates frozen do isolation later when ready but seems you have to switch to guanidine lysis not your curretly used trizol or phenol lysis.
Try LyseNow RNA kit or RNASound additives from Metammune, you can homogenize the tissue upfront and keep ltsates frozen do isolation later when ready but seems you have to switch to guanidine lysis not your curretly used trizol or phenol lysis.
#3
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Posted 17 December 2010 - 07:38 AM
It is snap frozen tissue taken from the animal during autopsy. The tissue is kept
in -80C.
in -80C.
