32RNA-sequencing is a popular next-generation sequencing technique for assaying 33 genome-wide gene expression profiles. Nonetheless, it is susceptible to biases that are 34 introduced by sample handling prior gene expression measurements. Two of the most 35 common methods for preserving samples in both field-based and laboratory conditions 36 are submersion in RNAlater and flash freezing in liquid nitrogen. Flash freezing in liquid 37 nitrogen can be impractical, particularly for field collections. RNAlater is a solution for 38 stabilizing tissue for longer-term storage as it rapidly permeates tissue to protect cellular 39RNA. In this study, we assessed genome-wide expression patterns in 30 day old fry 40 collected from the same brood at the same time point that were flash-frozen in liquid 41nitrogen and stored at -80°C or submerged and stored in RNAlater at room 42 temperature, simulating conditions of fieldwork. We show that sample storage is a 43 significant factor influencing observed differential gene expression. In particular, genes 44 with elevated GC content exhibit higher observed expression levels in liquid nitrogen 45 flash-freezing relative to RNAlater-storage. Further, genes with higher expression in 46RNAlater relative to liquid nitrogen experience disproportionate enrichment for 47 functional categories, many of which are involved in RNA processing. This suggests 48that RNAlater may elicit a physiological response that has the potential to bias biological 49interpretations of expression studies. The biases introduced to observed gene 50 expression arising from mimicking many field-based studies are substantial and should 51 not be ignored. 52 53