Sample variance can be introduced during many steps of sample preparation for an RNA-seq experiment. Sample variance arises because the data gathered in a sequencing run is only a fraction of the nucleic acid library. Technical variance arises because library preparation and sequence procedures use complex chemical reactions. Biological variance always exists because biological systems are complex and sensitive to any tampering.
It is therefore critically important to properly normalize RNA-seq data at multiple levels in order to make appropriate conclusions based on the final gene expression results.
Wadhwa RR, Azzam D. Variance. [Updated 2020 Sep 7]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK551689/