Selection of housekeeping gene for real time reverse transcription-PCR normalization in the hydrothermal vent shrimp Nautilocaris saintlaurentae

Abstract

Molecular biomarkers are considered as early warning signals of the biological impacts by any environmental changes. Therefore, we evaluated the stability of housekeeping genes (HKGs) in a hydrothermal vent shrimp, Nautilocaris saintlaurentae, to apply biomarker genes for monitoring any biological impacts by the future deep-sea mining activities. The shrimps were collected using suction samplers mounted on ROV (MAGNUM; Oceaneering International, Inc.) at a hydrothermal vent in Tofua Arc (1018m deep), the Southwest Pacific. Eight commonly used HKGs (α-tubulin, ß-actin, elongation factor1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L8 (RPL8), ribosomal protein S2 (RPS2), small subunit ribosomal RNA (SSU) and ubiquitin) were partially sequenced. Then primer pairs were specifically designed and tested their stability in RT-PCR assays. The expression of these potential reference genes was examined from two body parts (cephalothorax and abdomen) of individuals incubated at 4°C for 72 hours on board. The stability of HKGs was determined using Ct values and geNorm. As results, RPL8 and RPS2 were appeared to be the most reliable reference genes in all samples and these genes seem to be co-regulated (R2>0.87). However, each HKG was shown the different stability in each body part and thus the use of different HKGs was recommended to normalize expressed genes in differentae, to apply biomarker genes for monitoring any biological impacts by the future deep-sea mining activities. The shrimps were collected using suction samplers mounted on ROV (MAGNUM; Oceaneering International, Inc.) at a hydrothermal vent in Tofua Arc (1018m deep), the Southwest Pacific. Eight commonly used HKGs (α-tubulin, ß-actin, elongation factor1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L8 (RPL8), ribosomal protein S2 (RPS2), small subunit ribosomal RNA (SSU) and ubiquitin) were partially sequenced. Then primer pairs were specifically