Abstract
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.
Highlights
With advantages of relatively accurate quantification, high sensitivity and high throughput, quantitative reverse transcription polymerase chain reaction has become one of the most widely used techniques to detect changes in gene expression (Johnson et al, 2014; Phillips et al, 2009)
All tested reference gene amplification efficiencies were in the range of 90–110% (RPL28S (94.3%), ACTB (97.03%), TUBB (97.32%), CYC (96.61%), EF1α (92.03%), 18S ribosomal RNA (18S RNA) (95.54%) and Ubiq (100.89%)
(2) The results suggest that ACTB and TUBB are the most stable genes, with high expression levels when assessing salinity stress
Summary
With advantages of relatively accurate quantification, high sensitivity and high throughput, quantitative reverse transcription polymerase chain reaction (qRT-PCR) has become one of the most widely used techniques to detect changes in gene expression (Johnson et al, 2014; Phillips et al, 2009). There are inevitably a number of influencing factors that affect the efficiency of the reaction (Bustin et al, 2009), such as discrepancy in pipetting, the RNA quality and concentration, the efficiency of reverse transcription and amplification among different samples, the PCR procedures and primer amplification efficiency. An ideal reference gene should demonstrate a consistent level of expression across all tested tissues or conditions (Bustin & Bustin, 2002). There is increasing evidence that expression of assumed reference genes can vary observably with experimental conditions such as developmental stage and chemical treatment, significantly affecting relative quantification and qPCR result interpretation
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