Screening for qRT-PCR Internal Reference Genes in Clarias fuscus

Abstract

Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most widely used molecular techniques, which allows the detection and quantification of gene expression due to its high sensitivity, specificity, and reproducibility. To increase the reliability of the experimental results, internal reference genes are used to normalize the data. However, previous studies have shown that the stability of internal reference genes is influenced by experimental conditions and interspecies variations, and no universal internal reference genes have been identified for all species. Therefore, suitable internal reference genes must be identified for each species. The Hong Kong catfish (Clarias fuscus) has strong adaptability, high nutritional value, and tender flesh, and it was first introduced to large-scale aquaculture in the Guangdong and Guangxi provinces in the early 1970s. Currently, it is a key aquaculture species in the Guangdong-Guangxi region and one of the freshwater economic fish cultured in southern China. Thus, fairly extensive molecular biology and genetics studies of C. fuscus have been conducted, which in turn has increased the demand for quantitative gene expression analysis by qRT-PCR in these animals. Consequently, there is a growing demand for quantitative gene expression analysis by qRT-PCR in C. fuscus for various molecular biology and genetics studies. However, few studies have evaluated the internal reference genes for this species. Therefore, we aimed to identify suitable internal reference genes in different tissues and different stages of gonadal development of males and females to provide the necessary tools to support subsequent gene expression pattern analysis.In this study, we examined the expression of 13 internal reference genes (actb1, actb2, ef1a, eef1b2, rpl13a, rpl13, ccdc124, cfl1, nm23, eif3g, rap1a, ikbkg, and ywhab) in different stages of gonadal development and different tissues of adult fish of C. fuscus. We used four software tools, namely BestKeeper, GeNorm, NormFinder, and RefFinder, to evaluate the expression stability of the internal reference genes. BestKeeper software evaluates the stability of internal reference genes by calculating the standard deviation (SD) and coefficient of variation (CV). The results showed that in different tissues of adult fish, the stability ranking of these genes was as follows: actb1 > actb2 =ef1a > rpl13 > ywhab > eef1b2 > rpl13a > nm23 > eif3g > ccdc124 > rap1a > cfl1 > ikbkg. In different stages of male and female gonadal development, the stability ranking of these genes was as follows: actb2 > rpl13 > ef1a > rpl13a > actb1 > ywhab > cfl1 > rap1a > eef1b2 > eif3g > ccdc124 > ikbkg > nm23. NormFinder suggested that the best stability ranking was as follows: actb2=rpl13 > actb1 > rpl13a > ywhab > nm23 > rap1a > cfl1 > eif3g > ef1a > ccdc124 > ikbkg > eef1b2 in different tissues of adult fish and actb2=actb1 > rpl13 > ef1a > rpl13a > cfl1 > ywhab > rap1a > nm23 > eif3g > ccdc124 > ikbkg > eef1b2 in different stages of male and female gonadal development. The geNorm screens for the most stable internal reference genes by comparing the stability value (M) of each internal reference gene. It indicated that the final stability ranking in different tissues was actb2=rpl13 > rpl13a > actb1 > ef1a > ccdc124 > ywhab > rap1a > eif3g > nm23 > eef1b2 > ikbkg > cfl1, and actb1=rpl13 > actb2 > ef1a > rpl13a > cfl1 > ywhab > ccdc124 > rap1a > eif3g > nm23 > eef1b2 > ikbkg in different stages of male and female gonadal development. Finally, RefFinder analysis integrated the results of the other three software tools and showed that the comprehensive stability ranking of each gene in different tissues was actb2 > rpl13 > cfl1 > rap1a > ef1a > rpl13a > ikbkg > eif3g > nm23 > actb1 > eef1b2 > ywhab > ccdc124. In different stages of male and female gonadal development, the comprehensive stability ranking was actb2 > rpl13 > actb1 > cfl1 > rap1a > ef1a > rpl13a > ikbkg > eif3g > nm23 > eef1b2 > ywhab > ccdc124. Given this, we can conclude that actb2 is the best internal reference gene for C. fuscus. To validate the accuracy of the results, we selected actb2 and rpl13 as internal reference genes and compared their expression levels with those of four genes (zp3, atp2b3, slc13a5 and parp8) from transcriptome RNA-seq data. The fold changes in the expression levels were closer to the transcriptome data when using actb2 than when using rpl13, indicating that actb2 is more stable than rpl13.This study demonstrated that actb2 showed good stability among the 13 internal reference genes and could be used as an internal reference gene for different stages of gonadal development and different tissues of adult fish in C. fuscus. The results can provide technical support for the subsequent research on the functional gene expression patterns of C. fuscus and are expected to be applicable to other catfish species.

Keywords

• clarias fuscus
• qrt-pcr
• reference gene
• stability of expression