Forum:Is it okay to pool sample for RNAseq and proteomics study?

Question

[sha_6p]

Hi.

I'm about to do transcription and proteomics analysis for my study. But since I have few different time points, doing in replicates seems to be challenging on term of cost. I came across about pooling sample.

Is it okay to pool different few biological replicates together into 1 and proceed for sequencing? How reliable will that data be ?

Thank you in advance.

Answer 1

[andres.firrincieli]

Totally unreliable. Check other post in this forum " differential expression without replicates"

[Jianyu]

Agree, lack of enough replicates has been a problem for RNA-seq. Pooling sample only makes this thing worse

Answer 2

[h.mon]

You didn't explain in detail, but you implied pooling and leaving no replicates, which andres.firrincieli already rightly pointed is a no-go.

If you want to pool several samples, and use pools as biological replicates, this is also bad. Check the paper Experimental validation of methods for differential gene expression analysis and sample pooling in RNA-seq for details.

[jahnic.beck]

The paper suggests that pooling biological replicates delivers poor results but it does not mention anything about pooling wells containing the exact same cells. Since I'm using expensive experimental conditions, I would like to scale things down to a 24-well plate. However, to end up with a sufficient amount of cells I would need to pool two wells together for the RNA extraction. The wells, in this case, would not be biological replicate since the cells stem from the same culture. Does anyone here have experience with this kind of pooling?