RNA-Seq DE analysis - experimental planning

Key to a successful RNA-Seq differential expression analysis is good experimental planning. To help think about avoiding batch effects and separating our known sources of variation between sample groups, we are going choose the best way of batching the samples for our planned RNA-Seq experiment.

Our metadata contains information about our samples, including:

  • sample: name of sample
  • treatment: A or B
  • sex: M or F
  • replicate: 1, 2, 3, 4

Due to the lengthy process of tissue extraction, you can only isolate the RNA from 4 samples at the same time, and we have 4 biological replicates per group. Which of the following gives the best way of batching the samples for RNA Isolation to avoid batch effects:

  • Option A:


  • Option B:


  • Option C:


  • Option D:


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