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Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish
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Expressed repeat elements improve RT-qPCR normalization across a wide range of zebrafish gene expression studies
Suzanne Vanhauwaert1, Gert Van Peer1, Ali Rihani1
1Center of Medical Genetics, Ghent University, Ghent, Belgium.
Plos One
|October 14, 2014
Summary
Expressed repetitive elements (EREs) offer more stable reference targets for quantitative real-time PCR (RT-qPCR) normalization in zebrafish than traditional genes. This study identifies specific EREs as reliable candidates for accurate gene expression analysis.
Area of Science:
- Molecular Biology
- Genomics
- Zebrafish Model Organisms
Background:
- Accurate RT-qPCR data normalization relies on selecting stably expressed reference genes.
- Commonly used reference genes in zebrafish exhibit expression variability across diverse experimental conditions, potentially leading to unreliable conclusions.
Purpose of the Study:
- To evaluate expressed repetitive elements (EREs) as novel reference targets for RT-qPCR normalization in zebrafish.
- To compare the expression stability of EREs against commonly used reference genes under various experimental conditions.
Main Methods:
- Assessed expression stability of EREs and traditional reference genes across developmental time series, adult organs, and embryo treatments (morpholino, chemicals).
- Utilized geNorm and rank aggregation analysis to quantify and compare reference target stability.
Main Results:
- Expressed repetitive elements (EREs) demonstrated significantly higher overall expression stability compared to conventional reference genes.
- Identified a set of EREs (hatn10, dna15ta1, loopern4) exhibiting stable expression across a wide range of experimental settings.
Conclusions:
- EREs are superior reference targets for RT-qPCR normalization in zebrafish, offering improved data reliability.
- Proposed EREs (hatn10, dna15ta1, loopern4) are recommended for future zebrafish gene expression studies.
- The strategy for identifying and validating EREs holds potential for application in other species.
