ribosomal RNA depletion

[MLog]

Hello,
I'm using Epicentre Ribo-Zero kit for removal of rRNA from Drosophila. I checked RNA on Agilent, before and after depletion, and I observed a picture that seems strange for me (attached). A lot of small RNAs that do not seem to be present in the initial sample and nothing that looks like mRNA peak (it should be about 1000-2000 nt, I guess).
What is your opinion, is it normal?

[Olaf Blue]

Hi,

Drosophila is not the best substrate for Ribo-Zero, but it does work (see attached data). Not all rRNA species are readily removed, but if you do a Google Scholar search under "Ribo-Zero" and "Drosophila" there are at least one or two papers that cite this combination.

Here's an example:

Kumar et al. BMC Research Notes 2012, 5:230

I hope this is helpful.

Olaf

[weigrc]

Hello MLog,

How is the alignment result for above samples, I mean the % of sequencing reads mapped to rRNA?


Thanks for the sharing,
Wei

[MLog]

Hello MLog,

How is the alignment result for above samples, I mean the % of sequencing reads mapped to rRNA?


Thanks for the sharing,
Wei

Hi Wei,
not as good as I wish, actually - about 30-40%. Next time I did double depletion and it worked much better, with ~ 7% mapped ro rRNA.

[weigrc]

Hello MLog,

For the 1st depletion, do you think less input of the total RNA can help to reduce the 30-40% mapped rRNA??

What is your input amount of the total RNA?

Thanks,
Wei

[MLog]

Hello MLog,

For the 1st depletion, do you think less input of the total RNA can help to reduce the 30-40% mapped rRNA??

What is your input amount of the total RNA?

Thanks,
Wei

I usually take 1-3 micrograms of total RNA. With human and bacterial RNA it worked well - less than 1% of reads mapped to rRNA. Probably for Drosophila it's worth trying to take less.