[pmiguel]

I'm sorry if I was not clear. I have frozen cells isolated from adipose tissue that I store them at -80 freezer. I freeze cells without any buffer. I do nuclei isolation from 5 ml of frozen cells by adding same volume of lysis buffer.

I have used this isolated nuclei to get genomic DNA and it worked nicely but I'm not sure if this precedure is good to get good quality of RNA.

Typically it is RNAses in the cells that degrade RNA. Either that or allowing the pH to go high, especially in the presence of divalent cations.

So it is possible that during your nuclei isolation, the nuclear RNA is degrading.

I should add that I haven't actually seen a lot of RNA preps from nuclei. So I don't have any direct proof that nuclear total RNA has intact 18S and 28S RNA. Seems unlikely that it doesn't. So "degradation" is the most likely explanation.

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Phillip

[nucacidhunter]

Should I see the 18s and 28s peak when I running RNA BioAnalyser chips for Nuclei RNA? I don't see any peak in my run. Is that mean my nuclei RNA is degraded?

Visiting this web page https://norgenbiotek.com/display-product.php?ID=30 and clicking on "Application and Data" Tab will bring some gel images from nuclear and cytoplasmic RNA and it clearly shows presence of 18s and 28s in both RNA preps.

[pmiguel]

Visiting this web page https://norgenbiotek.com/display-product.php?ID=30 and clicking on "Application and Data" Tab will bring some gel images from nuclear and cytoplasmic RNA and it clearly shows presence of 18s and 28s in both RNA preps.

Thanks, that is useful. I always take images of that sort from vendors with a grain of salt. They have to deal with expectation -- if everyone expects two rRNA bands in a total RNA prep, they better be there.

But yes, the simplest answer is that cytoplasmic rRNA looks like nuclear rRNA.

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Phillip