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Biological replicates: individual samples or pooled samples for small RNA-seq?

Hi everyone,

I am planning a small RNA-seq experiment to identify miRNAs involved in plant–pathogen interaction. I have an infected treatment and a mock-inoculated control.

The issue is that the plant organs I inoculated were not all at exactly the same developmental stage. Some were slightly more advanced, while others were at an earlier stage. I am concerned that developmental variability could mask the infection signal, especially if some miRNAs are only induced in a subset of the infected samples.

I will sequence 4 biological replicates per condition. My question is: would it be better to use 4 individual organs as biological replicates, or 4 biological replicates consisting of pools of several organs each?

Any advice on the most appropriate design for differential miRNA expression analysis would be very helpful.

Thanks!

mirna-seq pooling
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updated by GenoMax GenoMax • written by Cristina Zamora • Cristina Zamora

If you pool several organs, will they come from the same plants, and then the biological replicates come from 4 separate plants?

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written by i.sudbery i.sudbery

The issue is that the plant organs I inoculated were not all at exactly the same developmental stage.

Are they different enough (e.g. days apart) that you already expect to see following?

I am concerned that developmental variability could mask the infection signal

Did you do matched samples (pre-innoculation and post)? If you have enough material to create individual libraries, you may be able to do paired comparisons and depending on the results could pool the sequence data afterwards, as biological replicates.

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written by GenoMax GenoMax

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