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Published on: May 31, 2014
Validation of reference genes for expression studies during craniofacial development in arctic charr
Ehsan Pashay Ahi1, Jóhannes Guðbrandsson, Kalina H Kapralova
1Institute of Life- and Environmental Sciences, University of Iceland, Reykjavik, Iceland.
Plos One
|June 21, 2013
Summary
This study identifies reliable reference genes for quantitative real-time PCR (qPCR) in Arctic charr morphs. These validated genes enable precise detection of gene expression differences in craniofacial development.
Area of Science:
- Evolutionary biology
- Genetics
- Developmental biology
Background:
- Arctic charr (Salvelinus alpinus) exhibit significant phenotypic polymorphism, with four distinct morphs in Lake Thingvallavatn, Iceland.
- Craniofacial differences among these morphs are evident during embryonic development, suggesting differential gene expression.
- Identifying stable reference genes is crucial for accurately quantifying gene expression in these morphs using quantitative real-time PCR (qPCR).
Purpose of the Study:
- To identify and validate stable reference genes for qPCR in Arctic charr morphs.
- To select reference genes capable of detecting subtle gene expression variations between morphs.
- To facilitate the study of differential gene expression in Arctic charr craniofacial development.
Main Methods:
- Twelve candidate reference genes were selected and their sequences identified from transcriptome data.
- Gene expression stability was assessed using qPCR and validated with statistical analyses and specialized software.
- Selected reference genes (ACTB, UB2L3, IF5A1) were tested in an expression study of craniofacial genes (sparc, mmp2, sox9) across different morphs and developmental stages.
Main Results:
- Many candidate genes showed stable expression, but their suitability as reference genes varied based on analytical methods.
- Three reference genes—ACTB, UB2L3, and IF5A1—were identified as highly consistent and suitable for qPCR in Arctic charr.
- Differential expression of sparc and mmp2 was detected in embryos of different Arctic charr morphotypes, validating the chosen reference genes.
Conclusions:
- ACTB, UB2L3, and IF5A1 are validated as reliable reference genes for qPCR in Arctic charr morphs.
- These reference genes enable robust detection of small relative expression changes during charr development.
- The study provides essential tools for investigating the genetic basis of craniofacial evolution in polymorphic Arctic charr.
