Experimental design and number of replicates for bulk RNA-Seq experiments

Please be aware that the number of samples per experimental group or condition in gene expression analyses are influenced by the following factors:

Biological and technical variation of replicates

The less homogeneous your biological replicates are, the more are you limited in finding statistical significantly expressed genes. This applies in particular to small differences in gene expression. Therefore rule of thumb: the smaller the differences you expext or the higher the sample to sample variability the more replicates you should do per experimental group.

Heterogeneity of sample quality

Differences in RNA integrity can introduce technical noise, thereby diminishing the sensitivity of detecting differentially expressed genes (DEGs). For bulk RNA-Seq we advise utilizing the total RNA-Seq workflow for samples of lower quality or greater heterogeneity. This approach is advantageous as it is not reliant on intact poly-A tails of mRNA, ensuring robustness in such challenging sample conditions.