Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation

Quantitative real-time reverse transcription-polymerase chain reaction(qRT-PCR) is a technique that is widely used for gene expression analysis,and its accuracy depends on the expression stability of the internal reference genes used as normalization factors.However,many applications of qRT-PCR used housekeeping genes as internal controls without validation.In this study,the expression stability of eight candidate reference genes in three tissues(intestine,respiratory tree,and muscle) of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm,NormFinder,delta CT,and RefFinder algorithms.The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation.In general,the P-tubulin(TUBB) gene was relatively stable in the intestine and respiratory tree tissues.The optimal reference gene combination for intestine was 40 S ribosomal protein S18(RPS18),TUBB,and NADH dehydrogenase(NADH);for respiratory tree,it was P-actin(ACTB),TUBB,and succinate dehydrogenase cytochrome B small subunit(SDHC);and for muscle it was a-tubulin(TUBA) and NADH dehydrogenase[ubiquinone]1 a subcomplex subunit 13(NDUFA13).These combinations of internal control genes should be considered for use in further studies of gene expression in A.japonicus during aestivation.

Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal reference genes used as normalization factors. However, many applications of qRT-PCR used housekeeping genes as internal controls without validation. In this study, the expression stability of eight candidate reference genes in three tissues （intestine, respiratory tree, and muscle） of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm, NormFinder, delta CT, and RefFinder algorithms. The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. In general, the β-tubulin （TUBB） gene was relatively stable in the intestine and respiratory tree tissues. The optimal reference gene combination for intestine was 40S ribosomal protein S18 （RPSI 8）, TUBB, and NADH dehydrogenase （NADH）; for respiratory tree, it was β-actin （ACTB）, TUBB, and succinate dehydrogenase cytochrome B small subunit （SDHC）; and for muscle it was α-tubulin （TUBA） and NADH dehydrogenase [ubiquinone] 1α subcomplex subunit 13 （NDUFA13）. These combinations of internal control genes should be considered for use in further studies of gene expression in A.japonicus during aestivation.