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Homogenize pancreas for RNA extraction


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3 replies to this topic

#1 wdyeo

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Posted 21 February 2013 - 03:02 AM

Dear All,
I am wondering, which is the best method to homogenize the pancreas of a mice to extract the RNA?
Should i use the whole pancreas or weigh it ~ 30mg per each reaction as suggested by the RNeasy Mini kit?
Thank you very much.

#2 jerryshelly1

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Posted 22 February 2013 - 06:51 AM

Someone in the lab has been doing something similar to your experiments. She has found that using either a small pestle and mortar or just chopping the tissue up with a razor blade in petri dish, works the best. I would definitely follow the protocol provided with your kit. If anything, do a quick google search against your kit and a variety of organs to see how they prepared there RNA. The biggest concern would probably be the duration of your emulsifier is left in solution with your pancreas. Don't use a homogenizer, RNA is delicate and easily destroyed.

#3 Doggie Science

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Posted 22 February 2013 - 08:15 AM

Hello!

I recommend you read a couple of technique papers on pancreas isolation. Due to the amount of RNAses present in the pancreas, isolating intact RNA from the pancreas is extremely difficult. In our lab, we do a quick resection (under 10 seconds) and snap freeze the tissue. We then pulvorize the tissue while frozen and hit with TRIZOL immediately.
There are other techinques that you can find on the web. You will have to find what works best for your lab setting. Just make sure to do Agilent for your RNA and calculate RIN to make sure you don't have degraded RNA.

#4 wdyeo

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Posted 23 February 2013 - 11:46 AM

Hi jerryshelly1 and doggie science,
Thanks alot guys for sharing your experiences and tips&tricks.
Have a nice day Posted Image





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