Enriching bacterial RNA from infection samples

[adam_ie]

Hi all,

I am working with RNA samples from in vitro infections of cells with bacteria. I would like to sequence the bacterial component of these samples. From a previous sequencing effort, I know that the vast majority (>99%) of the RNA comes from the host (eukaryotic) cells.

I previously attempted to deplete the eukaryotic and prokaryotic ribosomal RNA, as well as Poly-A transcripts, to leave only bacterial non-ribosomal RNA, but this did not prove sufficient and the vast majority of the RNA is still eukaryotic. Instead, I'm now considering the opposite strategy of enriching the bacterial RNA prior to sequencing.

From the data I have, I know that the amount of bacterial RNA that I actually want (i.e. non-ribosomal bacterial RNA) is in the range of ~1ng upwards per sample. One option that I am exploring to selectively enrich this RNA is the use of the SureSelect RNA Capture kit from Agilent (or a similar kit) using probes covering the whole genome of the bacterium.

I'm wondering if anyone has experience with a similar approach, or with enriching bacterial RNA from mixed samples generally? Will the SureSelect kit, or a similar kit, be able to handle an input as low as 1ng if it accounts for less than 1% of the sample?

Any thoughts or comments appreciated! Thanks

[nucacidhunter]

I previously attempted to deplete the eukaryotic and prokaryotic ribosomal RNA, as well as Poly-A transcripts, to leave only bacterial non-ribosomal RNA, but this did not prove sufficient and the vast majority of the RNA is still eukaryotic. Instead, I'm now considering the opposite strategy of enriching the bacterial RNA prior to sequencing.

I wonder if you have used this commercial product which follows a similar approach: http://www.lifetechnologies.com/au/e...tic-hosts.html

From the data I have, I know that the amount of bacterial RNA that I actually want (i.e. non-ribosomal bacterial RNA) is in the range of ~1ng upwards per sample. One option that I am exploring to selectively enrich this RNA is the use of the SureSelect RNA Capture kit from Agilent (or a similar kit) using probes covering the whole genome of the bacterium.

In theory this should work as long as you have enough RNA (mix of host and microbe) equivalent to mRNA input requirement of the kit.

[adam_ie]

I wonder if you have used this commercial product which follows a similar approach: http://www.lifetechnologies.com/au/e...tic-hosts.html

Yep, that's exactly the method I tried, without success. The proportion of eurkaryotic RNA seems to just be too high, even though it was considerably depleted with the above kit.

I'm now thinking that enrichment of the bacterial RNA is the only way to go in this case.