Identification of housekeeping gene for future studies exploring effects of cryopreservation on gene expression in...

Few studies have investigated the subcellular effects of low temperature on gene expression in shrimp and most other crustaceans. Before gene expression analysis is conducted, suitable housekeeping genes (HKGs) must be confirmed to account for differences in reverse transcription process efficiency among samples. Thus, this study aimed to verify five frequently used HKGs, namely 18S ribosomal RNA (18S rRNA), ATPase, histone 3, β-actin, and glyceraldehyde 3-phosphate dehydrogenase (gapdh) for use in experiments for assessing the molecular-scale effects of cryopreservation on coral banded shrimp (Stenopus hispidus) embryos. To conduct chilling studies, we subjected S. hispidus embryos to incubation at either 26 °C (control) or 5 °C for 0, 4, 8, 16, or 32 h. The software tools GeNorm, NormFinder, and Bestkeeper were employed to identify the most suitable HKG. GeNorm identified histone 3 and 18S rRNA as the most stable genes. By contrast, NormFinder determined that 18S rRNA is a stable gene for eye-formation and pre-hatch stage samples. Finally, Bestkeeper determined that gapdh and β-actin are the most suitable genes. This study is the first to identify suitable HKGs for studying shrimp embryos at low temperatures. Its findings can aid future research on evaluating the effects of cryopreservation on gene expression in crustaceans.