high rRNA after poly(A) selection

nboom · 10-24-2019, 12:59 AM

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 · 10-25-2019, 02:56 AM

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

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10-24-2019, 12:59 AM	#1
nboom Junior Member Location: London Join Date: Oct 2019 Posts: 1	high rRNA after poly(A) selection 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!

10-25-2019, 02:56 AM	#2
Michael.Ante Senior Member Location: Vienna Join Date: Oct 2011 Posts: 123	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