Identification and validation of the reference genes in the echiuran worm Urechis unicinctus based on transcriptome
Jiao Chen1, Yunjian Wang1, Zhi Yang2
1Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, China.
This study identifies novel, stable reference genes for accurate gene expression analysis in Urechis unicinctus using transcriptome data. New candidate genes proved more reliable than commonly used ones for RT-qPCR applications.
Area of Science:
- Molecular Biology
- Genomics
- Eukaryotic Gene Expression
Background:
- Real-time quantitative PCR (RT-qPCR) is vital for gene expression analysis.
- Accurate RT-qPCR relies on stable reference genes, which are often organism- or condition-specific.
- Transcriptome analysis offers a powerful approach for discovering novel reference genes.
Purpose of the Study:
- To identify and validate stable reference genes for RT-qPCR in the echiuran Urechis unicinctus.
- To leverage transcriptome data for reference gene discovery across different developmental stages and stress conditions.
- To compare the stability of newly identified reference genes against commonly used ones.
Main Methods:
- Transcriptome data analysis using the coefficient of variation (CV) method to identify candidate reference genes.
- Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses for functional annotation.
- Validation of candidate and common reference genes using RT-qPCR and stability analysis with geNorm, NormFinder, BestKeeper, and ∆Ct methods.
Main Results:
- Numerous candidate reference genes were identified across early development, adult tissues, and sulfide-stressed hindgut.
- Enrichment analyses revealed distinct functional roles for candidate genes in different conditions.
- Newly identified reference genes (STX12, EHMT1, LYAG) demonstrated superior stability compared to commonly used genes like ACTB.
- A significant negative correlation between transcriptome log2(TPM) and RT-qPCR Ct values was established, enabling Ct value estimation.
Conclusions:
- This research presents the first selection of reference genes for RT-qPCR from transcriptome data in Echiura.
- The identified novel reference genes provide a more reliable basis for gene expression studies in U. unicinctus.
- The findings offer crucial insights for future molecular and gene expression research in this organism.
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