Isolating microbial RNA

[Nullie]

I want to do check the microbial population in the gut before and after treatment, I isolated the total RNA from the intestine but how can I isolate the bacterial RNA from human RNA?
and after isolating how can I size select fragments of correct size to use for bridge amplification?

[JamieHeather]

You could use somekind of nucleic acid selection, like Agilent's SureSelect, but getting probes for a wide swathe of microbes might be a bit pricey if you're interested in more than one or two bugs.

Beyond that, I can't think of an easy way to separate them. You could deplete for host mRNA by selecting out 5' caps/polyA tails, but that'd still leave the bulk of the rRNAs and other non-mRNAs.

I imagine it would be easier to sort the bacteria from the cells by centrifugation before lysing - that said I'm no expert on microbes or GI stuff, there might well be some feature of microbial RNA that I haven't remembered that you could use!

You can do size selection by gel excision, or using an e-gel or something similar.

[ZWB]

We've developed a technique that separates host transcripts from bacterial transcripts, but right now it only works if the organism of interest is culturable. You may be able to make probes using the technique described against a consortium of bacteria that are found in the gut as well.

http://www.ncbi.nlm.nih.gov/pubmed/23535274

[JamieHeather]

Actually that's a great idea - you could just do cDNA subtraction using human cDNA from a sample that won't have any microbes (or at least none of the same as what you'd expect from a gut sample), leaving you with ideally just the gut-specific microbe sequences.

This means you're selecting out what you don't want (i.e. human) rather than what you do (bug).

[ZWB]

Jamie,

I've tried that before and it works fairly well. We called it negative capture for the reason you described (capturing what you don't want). The problem is getting the most similar host RNA to your experimental condition. If the transcriptional profile of the probes differs greatly from the experimental condition, the capture is largely ineffective. Also we find that the simpler the genome the better capture works. Our best results are in viruses then bacteria and then eukaryotes.

Zach

[JamieHeather]

That makes sense. In all honesty I've never done it, I just know it from the historical immunology literature; it was used heavily to clone T-cell receptor sequences before we knew what they looked like, just by subtracting B-cell libraries.

I think they used to have to do a number of iterations of subtraction (which I guess reflects the differences between the T-cell and B-cell transcriptomes).

I suppose the very best subtraction library would be the exact same tissue, minus any bugs - maybe by culturing samples with heavy antibiotic selection? Although I presume too long in culture would also alter the transcription profiles.

I wonder if you could just try a few rounds of broad human-sequence library? So make a huge library from as many different cell types as you could, particularly as many gut related types, and use that? Presumably there'll come a point where the subtracting library is saturating with regards to what's in the sample to be sequenced - I suppose it's just a trade off whether this is reached before you lose all of your target microbial stuff.

[Nullie]

is it possible to isolate 16srRNA from the total RNA sample?

[ZWB]

It's harder not to isolate 16S rRNA from a sample. >90% of the bacterial transcripts will be rRNA. You probably don't need to do anything special to get rRNA, although you could make 16S probes to pull the transcripts out of the pool.