Ribo-zero bacteria vanishing RNA

[micro1510]

Hello,

I'm trying to perform rRNA depletion on my total extracted RNA from a panel of E. coli isolates using the Ribo-Zero bacteria kit but am getting extremely low recoverable yields (around 1-2 ng total RNA). I've been informed I need 10 ng of RNA in 5 µl total volume for library prep for RNA-seq. I've run the before and after samples on the bioanalyser and it seems fine before performing rRNA depletion. I've tried all the Illumina recommended post depletion purification methods (ETOH ppt, Qiagen mini-elute and clean up and zymo clean and concentrator columns) but sadly this seems to be as good as it gets.

Any advice would be greatly appreciated!

Thanks

[jteeee2]

I have also seen considerably low yields post Ribo-Zero for bacterial samples. Have you tried simply proceeding into library prep with the material you have? The success of this strategy might depend on the kit manufacturer you are using, but I can tell you that I have recently proceeded into the Kapa Biosystems RNA-Seq kit using a low amount of RNA (I can't tell you the exact mass because I couldn't detect it on Qubit with 1 uL input) and generated successful libraries. It might be worth a shot with one of your samples. Best of luck!

[ScienceGrrl]

I had the opposite problem in my previous position of too much sample at the end b/c of failure to remove rRNA. I tested my depletion using Illumina's suggestion of running qPCR assay using GAPDH and 18S to see the amount of material before and after depletion. If your non-rRNA species are low in the pre depletion, you may be getting all you can.

[kerplunk412]

Have you tried inputting more total RNA into the ribo-depletion, or do you have limited amounts?

Also, 1-2 ng should be fine for many RNA-Seq library prep protocols, so you may want to look into an alternative protocol; there are lots of companies making RNA-Seq kits these days. Feel free to PM me for more information in Bioo Scientific's offerings. As a disclaimer, I work for Bioo Scientific.

[micro1510]

Thanks for all the replies everyone, much appreciated! The library preps are being performed at our universities genomics facility and they are so far being rather stubborn about the required amount of RNA, however now that people have mentioned they have had success with lower amounts I am attempting to convince them to give it a go. I am currently putting in the maximum recommended amount of RNA into the Ribo-zero kit, however I have been considering increasing it past the recommended values as I have a huge amount of total RNA and am currently still getting complete rRNA depletion.

Thanks again

[kerplunk412]

Definitely use more starting RNA if you can. Worst-case scenario is you have more rRNA reads, but a library with high rRNA contamination is better than no library at all.

5 uL is also a pretty low input volume for RNA-Seq, I think typical kits are more like 15 uL. If your facility accepts completed libraries you may want to think about doing the preps on your own so you could have a little more flexibility with input amount and volume. This would also probably be cheaper, and would help you learn more about the library prep process. Also, the Ribo-Zero is probably one of the most expensive parts of your sequencing project, so it would be nice to be able to use the samples you have already prepared.