How can I go for RNA pooling of different tissues for Transcriptome Analysis?

Question

[snrajainsect]

I am interested in RNA seq analysis with egg, larvae, pupa and adult of lepidopteran insect with this I would like to know how to prepare the pooled RNA sample for RNA seq analysis. In addition I wonder the total number of replications to be maintained for effective result.

[Fabio Marroni]

Hi, I am not sure in biostars is the best place to ask this question. This site is about bioinformatics. You might try asking the question on seqanswers or other forums related to molecular biology experiments.

Answer 1

[h.mon]

If possible, do not pool individuals, try to extract RNA from single individuals, and prepare each library separately and with a different barcode. For transcriptome assembly (if thats the case), you can pool all the reads and assemble.

As for the number of biological replicates, three per treatment is the bare minimum, but with only three you will have low statistical power for differential gene expression analysis. Keep in mind that is not uncommon to have "bad" samples (see Gierlinski et al., 2015), so to have 3 biological replicates, often you have to sequence more than 3 per treatment.

Schurch et al. (2016) is a very detailed analysis of RNAseq sample sizes and statistical power, according to their recommendations, 6 biological replicates per condition should be considered the minimum for most experiments.