no detectable RNA after rRNA depletion

gretagecse · 07-08-2019, 11:58 PM

Hi all,

I am working with bacterial RNAseq, and like for many others, the discontinuation of Ribo zero made my life a bit more challenging.

I have been testing an alternative from siTOOLS, called riboPOOLS, but so far I haven't been lucky with it. They are using the oligo method with dynabeads, similar to Ribo zero.

My input RNA is around 2-4 µg, within the range that they require. And I follow every step in the protocol, correctly. However, I am having 0 RNA after the depletion. I tried to measure with Quibit HS RNA assay and Bioanalyzer pico chip, and there is nothing left there.

Before depletion, I can see small and mRNAs from the Bioanalyzer results, but they are gone after the depletion.
I heard about very low concentration of mRNA after rRNA depletion, which makes sense due to the high abundance of rRNA, but I am having 0 mRNA or sRNA after the depletion.

I also did a control experiment, where I lowered the concentration of the oligos they suggest, and after that depletion, I had RNA outcome. This convinces me that I am not making a mistake in the protocol.

Has anyone experienced such a thing? Maybe with the Ribo zero before?

Cheers,
Greta

furor · 09-17-2019, 01:12 AM

Hello,

were you able to solve this? I have heard positive news about the pan-prokaryotic kit from the university sequencing center here, and I would be interested in using a combination riboPOOL.

Cheers

nucacidhunter · 09-17-2019, 07:56 PM

RiboFree total RNA-seq library prep from Zymo Research looks good on paper and does not require species specific depletion step but I have not tried yet:

https://www.zymoresearch.com/product...na-library-kit

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07-08-2019, 11:58 PM	#1
gretagecse Junior Member Location: Denmark Join Date: May 2019 Posts: 3	no detectable RNA after rRNA depletion Hi all, I am working with bacterial RNAseq, and like for many others, the discontinuation of Ribo zero made my life a bit more challenging. I have been testing an alternative from siTOOLS, called riboPOOLS, but so far I haven't been lucky with it. They are using the oligo method with dynabeads, similar to Ribo zero. My input RNA is around 2-4 µg, within the range that they require. And I follow every step in the protocol, correctly. However, I am having 0 RNA after the depletion. I tried to measure with Quibit HS RNA assay and Bioanalyzer pico chip, and there is nothing left there. Before depletion, I can see small and mRNAs from the Bioanalyzer results, but they are gone after the depletion. I heard about very low concentration of mRNA after rRNA depletion, which makes sense due to the high abundance of rRNA, but I am having 0 mRNA or sRNA after the depletion. I also did a control experiment, where I lowered the concentration of the oligos they suggest, and after that depletion, I had RNA outcome. This convinces me that I am not making a mistake in the protocol. Has anyone experienced such a thing? Maybe with the Ribo zero before? Cheers, Greta

09-17-2019, 01:12 AM	#2
furor Member Location: Europe Join Date: Jan 2012 Posts: 10	Hello, were you able to solve this? I have heard positive news about the pan-prokaryotic kit from the university sequencing center here, and I would be interested in using a combination riboPOOL. Cheers

09-17-2019, 07:56 PM	#3
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	RiboFree total RNA-seq library prep from Zymo Research looks good on paper and does not require species specific depletion step but I have not tried yet: https://www.zymoresearch.com/product...na-library-kit