Selection of housekeeping gene for quantitative RT-PCR normalization in the hydrothermal vent shrimp Nautilocaris saintlaurentae

Abstract

Deep-sea vent invertebrates are known to tolerate a wide range of temperatures and pressures but a few studies have been conducted the analysis of relative genes expression effected on environmental stresses in vent organisms. A vent shrimp Nautilocaris saintlaurentae was collected by ROV named MAGNUM (Oceaneering International, Inc.) at Tofua Arc (depth 1018m) in the Southwest Pacific on 15 April 2011. Before genes expression experiments were assessed using the real-time qRT-PCR for evaluating physiological status of N. saintlaurentae, it was carefully evaluated whether a certain housekeeping gene (HKG) is stably expressed in the experimental system under study. Five commonly used HKGs (small subunit ribosomal RNA (SSU), ribosomal protein isoform 8 (RPL8), ribosomal protein S2 (RPS2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ß-actin) were partially sequenced in N. saintlaurentae. Then PCR primer pairs were specifically designed and tested in real-time qRT-PCR assays. The expression of these potential reference genes was examined in 12 samples from six individuals that incubated at 4°C for 96 hours in-situ. The stability of five HKGs was determined using Ct mean value and geNorm software. RPL8 and RPS2 appeared as the most reliable reference genes with low M values (less than 0.4) for the normalization of qRT-PCR data in N. saintlaurentae.rimp Nautilocaris saintlaurentae was collected by ROV named MAGNUM (Oceaneering International, Inc.) at Tofua Arc (depth 1018m) in the Southwest Pacific on 15 April 2011. Before genes expression experiments were assessed using the real-time qRT-PCR for evaluating physiological status of N. saintlaurentae, it was carefully evaluated whether a certain housekeeping gene (HKG) is stably expressed in the experimental system under study. Five commonly used HKGs (small subunit ribosomal RNA (SSU), ribosomal protein isoform 8 (RPL8), ribosomal protein S2 (RPS2), glyceraldehyde-3-phospha