Abstract
Reference genes used in normalizing qRT-PCR data are critical for the accuracy of gene expression analysis. However, many traditional reference genes used in zebrafish early development are not appropriate because of their variable expression levels during embryogenesis. In the present study, we used our previous RNA-Seq dataset to identify novel reference genes suitable for gene expression analysis during zebrafish early developmental stages. We first selected 197 most stably expressed genes from an RNA-Seq dataset (29,291 genes in total), according to the ratio of their maximum to minimum RPKM values. Among the 197 genes, 4 genes with moderate expression levels and the least variation throughout 9 developmental stages were identified as candidate reference genes. Using four independent statistical algorithms (delta-CT, geNorm, BestKeeper and NormFinder), the stability of qRT-PCR expression of these candidates was then evaluated and compared to that of actb1 and actb2, two commonly used zebrafish reference genes. Stability rankings showed that two genes, namely mobk13 (mob4) and lsm12b, were more stable than actb1 and actb2 in most cases. To further test the suitability of mobk13 and lsm12b as novel reference genes, they were used to normalize three well-studied target genes. The results showed that mobk13 and lsm12b were more suitable than actb1 and actb2 with respect to zebrafish early development. We recommend mobk13 and lsm12b as new optimal reference genes for zebrafish qRT-PCR analysis during embryogenesis and early larval stages.
Highlights
Quantitative real-time polymerase chain reaction has been widely employed for gene expression analysis because of its specificity, sensitivity and reproducibility [1, 2]
Searches for reference genes in zebrafish are generally based on identification of orthologues of genes stably expressed in mammalian tissue, mainly from mice or humans [12], such as β2-microglobulin [13], gapdh [16] and β-actin [17]
Since ideal reference genes for zebrafish developmental studies should be moderately or highly expressed at different developmental stages, the minimum Reads per kilobase per million (RPKM) value was set to 40 (>40) and it acted as a major parameter for the round of screening
Summary
Quantitative real-time polymerase chain reaction (qRT-PCR) has been widely employed for gene expression analysis because of its specificity, sensitivity and reproducibility [1, 2]. To obtain precise results from qRT-PCR assays related to zebrafish development, a reliable normalization reference gene should be used that is expressed stably with minimal variation in expression levels. The qRT-PCR expression stabilities of the candidates were further evaluated using four statistical algorithms, namely delta-CT [33], geNorm [34], BestKeeper [35] and NormFinder [36], and compared to those of the commonly used zebrafish reference genes actb and actb. The qRT-PCR expression stabilities of the candidates were further evaluated using four statistical algorithms, namely delta-CT [33], geNorm [34], BestKeeper [35] and NormFinder [36], and compared to those of the commonly used zebrafish reference genes actb and actb2 We used the newly selected reference genes to normalize three well-studied target genes during zebrafish embryogenesis, thereby demonstrating their effective use
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