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Microarray-based uncovering reference genes for quantitative real time PCR in grapevine under abiotic stress
João L Coito1, Margarida Rocheta, Luísa Carvalho
1Centro de Botânica Aplicado à Agricultura, Departamento de Recursos Naturais, Ambiente e Território, Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Lisbon, Portugal.
Selecting stable reference genes is crucial for accurate gene expression analysis using quantitative real time polymerase chain reaction (RT-qPCR). This study validates grapevine reference genes under various stress conditions, finding multiple combinations suitable for normalization.
Area of Science:
- Plant molecular biology
- Gene expression analysis
- Biotechnology
Background:
- Quantitative real time polymerase chain reaction (RT-qPCR) is a key technique for mRNA detection and transcription analysis.
- Accurate RT-qPCR data relies on proper normalization using stable reference genes.
- Microarray data can aid in selecting suitable reference genes for specific experimental conditions.
Purpose of the Study:
- To identify and validate stable reference genes for RT-qPCR in grapevine under water and heat stress.
- To assess the suitability of selected reference genes for normalizing gene expression data in stress marker genes.
Main Methods:
- Selection of candidate reference genes (PADCP, ubiq, TIF, TIF-GTP, VH1-IK, aladin-related, act, L2) based on grapevine microarray data.
- Validation of gene stability using RT-qPCR in grapevine leaves under field and greenhouse conditions (water stress, heat stress, combined stress).
- Analysis of gene stability using GeNorm, NormFinder, and the ΔCq method, followed by testing in stress marker genes.
Main Results:
- Several combinations of reference genes were identified as suitable for data normalization across different stress conditions.
- Stability analysis revealed that different methods suggested varying optimal gene combinations.
- Normalization of stress marker genes (PCO, Galsynt, BKCoAS, HSP17) did not significantly differ when using the identified reference gene combinations.
Conclusions:
- Multiple reference gene combinations are suitable for normalizing RT-qPCR data in grapevine under water and heat stress.
- The choice of reference gene combination did not significantly impact the expression analysis of tested stress marker genes.
- Microarray analysis is a valuable tool for selecting appropriate reference genes for RT-qPCR in grapevine.
