I have been trying to extract RNA from mouse spleen for a while and the results it's always the same, I end it up with the whole RNA degraded. I use the RNA-II extraction kit from Macherey-Nagel and the amount and the 260/280 - 260/230 ratios are quite good but when I run my samples in a gel, I can't get to see the ribosomic RNA bands that indicate quality. However most of other tissues such as heart, kidney, muscle, ovary, lungs and brain are working perfectly fine. I don't know what I am doing wrong or what should I do differently with this. I hope some of you could give me your feedback.
Thank you in advance.
Spleen is notoriously rich in nucleases, so the spleen needs to be homogenized immediately in the lysis/chaotropic agent immediately after dissection/removal from the animal. Do not wait at all between the time the spleen is removed and the time it is homogenized in whatever nuclease-inactivating solution you are using. A good link for supportive info can be found here:
https://www.thermofisher.com/us/en/home/references/ambion-tech-support/rna-isolation/tech-notes/isolation-of-total-rna-from-difficult-tissues.html
Spleen is notoriously rich in nucleases, so the spleen needs to be homogenized immediately in the lysis/chaotropic agent immediately after dissection/removal from the animal. Do not wait at all between the time the spleen is removed and the time it is homogenized in whatever nuclease-inactivating solution you are using. A good link for supportive info can be found here:
https://www.thermofisher.com/us/en/home/references/ambion-tech-support/rna-isolation/tech-notes/isolation-of-total-rna-from-difficult-tissues.html
I concur. I will only add that to minimise degradation dome persons snap freeze tissue in liquid nitrogen and the proceed immediately to homogenisation in buffer
Thank you very much guys, I will definitely try these tips!
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