Problem with RNA seq library preparation

Question

[sri]

Is it necessary to extract RNA from all samples (replicates) on the same day?

I am working with an RNA binding protein in Ustilago maydis. Deletion of the RBP cause aberrant cell morphology in the organism. I want to perform a differential gene expression analysis between wildtype and the RBP mutant U.maydis strain. a few days back, I sent my RNA samples for sequencing. The design of experiment is, two groups (wildtype vs RBP knock out mutant) and 3 replicates for each group. My problem is out of the 6 samples, The 3rd replicate in both the wild-type and mutant sample was prepared completely on a different day (but the experimental condition was the same!!). I am so worried what if this two sample will cause a major problem in my data analysis??

Answer 1

[Devon Ryan]

Is it necessary to extract RNA from all samples (replicates) on the same day?

As much as possible, yes, you should try to perform RNA extractions on the same day. I've been bitten by not doing this before, where the day I performed the extraction ended up being a significant batch effect (presumably I was slow one day so some samples were a bit degraded). Now just because problems like this can happen doesn't mean that they'll happen to you or that the effect will be large enough to worry about. If you don't see any clustering by day on a PCA plot then you got lucky :)