23 months ago by
So with 40 samples you may be able to bring down library prep costs with a high throughput protocol. Usually this means you barcode your individual samples with some sort of in line barcode early on and then you just do one library prep and sequence it. We did this with the RNA Tag Seq protocol for prokaryotic RNA and it is a lot cheaper.
This is one for miRNA but I believe there are others:
I don't know if anyone is offering this commercially as a service or kit. You may want to check around.
If that seems too daunting (and it is of course pricey to use a protocol you don't know well):
More reps with shallow sequencing is usually better than fewer reps with deeper sequencing. If you could reduce the total cost by doing minimal sequencing on the 40 reps I would do that. Of course, the library prep kits are expensive.
If you must pool, I would think pooling the RNA would allow for better quants to balance the pools than pooling biomaterial, i.e. ensuring there is even pooling between samples and one is not dominating the pool.
There are some older papers that say pooling is theoretically fine. People used to pool a lot with mircroarrays because the arrays themselves are so expensive. But the problem with pooling is that outlier samples may be dominating your findings but these become invisible in the sequencing. So if there is expected to be diversity among individuals (or if there is unexpected diversity) in your disease state then you may get garbage out of the experiment.
If there isn't diversity and you get truth you may nevertheless have a reviewer who expects diversity and doesn't believe your (true) paper. Then you're stuck.
In any case, I would have at least 3 pools per disease, so you can at least identify outlier pools if not outlier samples.
If you are only interested in big fold changes you may be able to get that with some individual reps. For regular RNA Seq 3 reps usually gets you most of your 4x fold changes and some of your 3X. I don't know for miRNA.