high rRNA after poly(A) selection

[nboom]

Hi,

About half our samples post library prep (poly(A) selection) + sequencing had around 50% rRNA content. The samples were re-prepped in the facility but the rRNA contents were similar a second time.

The facility now argue that the rRNA "contamination" is sample related and not due to failed library prep. Since the poly(A) selection enriches for all RNAs that have a poly(A) tail, mostly mRNAs, rRNA removal should have been effective? What can have gone wrong?

The tissue is skin and we used mirVana kit to extract RNA (perhaps not optimal - could the inclusion of small RNAs caused an issue in the library prep?). All our samples had RIN of 8 and above.

Thanks in advance!

[Michael.Ante]

Hi,

What library prep have you used?
Do you differentiate between MT-rRNA and nucleic rRNA? The mitochondrial rRNA molecules have a polyA tail and are therefore detectable after polyA selection.

Cheers,

Michael