Has anyone noticed a higher than expected rRNA ratio for mammalian cells on a RNA Nano chip? I've attached the bioanalyser printout. Of my 6 samples, my minimum is 2.7 and highest is 5! It appears that for samples with a rRNA ratio over 3.2, the Expert 2100 gives "error: 4501, unexpected sample type" and cannot calculate RIN.
I extracted RNA from a murine spermatogonial stem cell line using Trizol and the Zymo Direct-zol on column kit. I performed on-column DNase digestion.
Does anyone know what caused this high rRNA ratio? Alternatively, does anyone know if these samples are suitable for an Affy gene array?
I plan to run these samples on an Affy mouse gene 2.0 ST array.
Update: These samples were run on Affy arrays without any problems.
Hi Vavind,
it seems to me your RNA's are clearly not degraded, so that's good, and that's what you want to know for your bioanalyzer run; so with that in mind you could say it's safe to run.
I'm assuming you checked whether your specific cell type does not have a tendency to show odd rRNA ratio's? (I have no experience with this cell type)
Is it perhaps possible that you accidentally "enritched" for large RNA? (for example, if you do a precipitation in isopropanol and fish out the RNA, you are enritching for larger molecular, as they are more likely to get tangled in the web then smaller molecules?)
If that is so, this would also bias your gene expression outputs...
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