To Pool or not to Pool?

[Ayeh]

Hi

I am PhD candidate of animal genetic. My thesis is “Comparison of immune system genes (GE) pathway of local and commercial poultry by transcriptome analysis”. I have a question, pool sample or individual sample is better?

[dpryan]

Don't pool

[melis]

I would suggest not to ppol your samples but do a multiplexed library. Good luck!

[Skiaphrene]

Don't pool

Then again you may not have a choice: if you are extracting RNA from a very small cell population within your organism (chicken?) and not getting enough RNA to actually do the sequencing on (and you are not prepared to do amplification for some reason). Is this what you are doing?

If so, it certainly has been done before - immunologists do pool sorted immune cells from multiple mice to have enough RNA for sequencing (sorry I don't have a reference of the top of my head, but I have seen it. As you might expect it isn't something they put forwards much!) (though one can easily argue that inter-mice variability is much less an issue for laboratory strains than for local & commercial poultry!).

Then again, if you think about it, extracting RNA from multiple cells is already a type of pool... it all depends on what level you're prepared to deal with biological variability.

And if I misinterpreted your question, sorry!


Best,

-- Alex

[Jeremy]

Transcriptome sequencing for differential expression is basically high throughput qPCR. You can't get qPCR published without biological replicates, if you tried to published qPCR with a single pool of cases and a single pool of controls you would have a very, very bad time.

[dpryan]

@Jeremy: Nicely put!