Dataset Information

Identification of Novel Reference Genes Suitable for qRT-PCR Normalization with Respect to the Zebrafish Developmental Stage.

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.

SUBMITTER: Hu Y

PROVIDER: S-EPMC4758726 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Identification of Novel Reference Genes Suitable for qRT-PCR Normalization with Respect to the Zebrafish Developmental Stage.

Hu Yu Y Xie Shuying S Yao Jihua J

PloS one 20160218 2

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 fr ...[more]

PMID: 26891128

Similar Datasets

Project description:BackgroundDendrocalamus latiflorus Munro distributes widely in subtropical areas and plays vital roles as valuable natural resources. The transcriptome sequencing for D. latiflorus Munro has been performed and numerous genes especially those predicted to be unique to D. latiflorus Munro were revealed. qRT-PCR has become a feasible approach to uncover gene expression profiling, and the accuracy and reliability of the results obtained depends upon the proper selection of stable reference genes for accurate normalization. Therefore, a set of suitable internal controls should be validated for D. latiflorus Munro.ResultsIn this report, twelve candidate reference genes were selected and the assessment of gene expression stability was performed in ten tissue samples and four leaf samples from seedlings and anther-regenerated plants of different ploidy. The PCR amplification efficiency was estimated, and the candidate genes were ranked according to their expression stability using three software packages: geNorm, NormFinder and Bestkeeper. GAPDH and EF1α were characterized to be the most stable genes among different tissues or in all the sample pools, while CYP showed low expression stability. RPL3 had the optimal performance among four leaf samples. The application of verified reference genes was illustrated by analyzing ferritin and laccase expression profiles among different experimental sets. The analysis revealed the biological variation in ferritin and laccase transcript expression among the tissues studied and the individual plants.ConclusionsgeNorm, NormFinder, and BestKeeper analyses recommended different suitable reference gene(s) for normalization according to the experimental sets. GAPDH and EF1α had the highest expression stability across different tissues and RPL3 for the other sample set. This study emphasizes the importance of validating superior reference genes for qRT-PCR analysis to accurately normalize gene expression of D. latiflorus Munro.

Dataset Information

Identification of Novel Reference Genes Suitable for qRT-PCR Normalization with Respect to the Zebrafish Developmental Stage.

Publications

Identification of Novel Reference Genes Suitable for qRT-PCR Normalization with Respect to the Zebrafish Developmental Stage.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets