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Selection and Validation of Reference Genes for qRT-PCR Analysis in Neocinnamomum caudatum
Yi Gan1, Haoyang Geng1, Yuanlin Zhang1
1College of Advanced Agricultural Sciences, Zhejiang A&F University, Hangzhou 311300, China.
Plants (Basel, Switzerland)
|July 15, 2026
Summary
This study identifies the best reference genes for accurate gene expression analysis in the woody oil plant Neocinnamomum caudatum. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is recommended for general use, while ACT11 and TUA are suitable for specific tissues like leaves, flowers, and developing seeds, respectively.
Area of Science:
- Plant Molecular Biology
- Gene Expression Analysis
- Biotechnology
Background:
- Neocinnamomum caudatum is a woody oil plant with seeds rich in valuable fatty acids.
- Investigating lipid metabolism in N. caudatum requires reliable quantitative real-time PCR (RT-qPCR).
- Validated reference genes are crucial for accurate RT-qPCR but are currently lacking for N. caudatum.
Purpose of the Study:
- To systematically evaluate the expression stability of seven candidate reference genes in N. caudatum.
- To identify the most suitable reference genes for RT-qPCR normalization across different tissues (leaves, flowers, developing seeds).
- To provide a foundation for future molecular studies on lipid biosynthesis in N. caudatum.
Main Methods:
- Seven candidate reference genes (EF-1α, ACT2, ACT11, UBQ11, TUA, F-BOX, GAPDH) were selected based on transcriptomic data.
- Expression stability was analyzed in leaves, flowers, and developing seeds using geNorm, NormFinder, and BestKeeper algorithms.
- RankAggreg was used for comprehensive ranking, and expression of NcFAD2 and NcFatB genes validated the findings.
Main Results:
- GAPDH was the most stable reference gene across all tested tissues.
- ACT11 was the most stable in leaf and flower samples, while TUA was most stable in developing seeds.
- EF-1α and TUA (in leaves/flowers) were identified as the least stable and unsuitable for normalization.
Conclusions:
- This study provides the first systematic validation of reference genes for RT-qPCR in N. caudatum.
- Recommended reference genes (GAPDH, ACT11, TUA) enable accurate gene expression studies in specific tissues.
- The findings support future research into the molecular mechanisms of lipid metabolism in this underutilized plant.
