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Real-time quantitative reverse transcription-PCR (qRT-PCR) is an undeniably effective tool for measuring levels of gene expression, but the accuracy and reliability of the statistical data obtained depend mainly on the basal expression of selected housekeeping genes in many samples. To date, there have been few analyses of stable housekeeping genes in  Onchidium reevesii  under salinity stress and injury. In this study, the gene expression stabilities of seven commonly used housekeeping genes,  CYC ,  RPL28S ,  ACTB ,  TUBB ,  EF1a ,  Ubiq  and  18S RNA , were investigated using BestKeeper, geNorm, NormFinder and RefFinfer. Although the results of the four programs varied to some extent, in general,  RPL28S ,  TUBB ,  ACTB  and  EF1a  were ranked highly.  ACTB  and  TUBB  were found to be the most stable housekeeping genes under salinity stress, and  EF1a  plus  TUBB  was the most stable combination under injury stress. When analysing target gene expression in different tissues,  RPL28S  or  EF1a  should be selected as the reference gene according to the level of target gene expression. Under extreme environmental stress (salinity) conditions,  ACTB  (0 ppt, 5 ppt, 15 ppt, 25 ppt) and  TUBB  (35 ppt) are reasonable reference gene choices when expression stability and abundance are considered. Under conditions of 15 ppt salinity and injury stress, our results showed that the best two-gene combination was  TUBB  plus  EF1a . Therefore, we suggest that  RPL28S ,  ACTB  and  TUBB  are suitable reference genes for evaluating mRNA transcript levels. Based on candidate gene expression analysis, the tolerance of  O. reevesii  to low salinity (low osmotic pressure) is reduced compared to its tolerance to high salinity (high osmotic pressure). These findings will help researchers obtain accurate results in future quantitative gene expression analyses of  O. reevesii  under other stress conditions.

Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury in Onchidium reevesii