PUBLICATION
Identification of Novel Reference Genes Suitable for qRT-PCR Normalization with Respect to the Zebrafish Developmental Stage
- Authors
- Hu, Y., Xie, S., Yao, J.
- ID
- ZDB-PUB-160219-3
- Date
- 2016
- Source
- PLoS One 11: e0149277 (Journal)
- Registered Authors
- Yao, Jihua
- Keywords
- Zebrafish, Gene expression, Embryos, Polymerase chain reaction, Complementary DNA, Embryogenesis, Pseudogenes, RNA extraction
- Datasets
- GEO:GSE45706
- MeSH Terms
-
- Animals
- Cluster Analysis
- Gene Expression Profiling*
- Gene Expression Regulation, Developmental*
- High-Throughput Nucleotide Sequencing
- RNA Stability
- Real-Time Polymerase Chain Reaction/standards*
- Reference Standards
- Transcriptome
- Zebrafish/embryology*
- Zebrafish/genetics*
- PubMed
- 26891128 Full text @ PLoS One
Citation
Hu, Y., Xie, S., Yao, J. (2016) Identification of Novel Reference Genes Suitable for qRT-PCR Normalization with Respect to the Zebrafish Developmental Stage. PLoS One. 11:e0149277.
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping