Selection and Validation of the Most Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Salvia...

Area of Science:

• Plant Molecular Biology
• Pharmacognosy
• Biotechnology

Background:

• Rosemary (Salvia rosmarinus) possesses significant pharmacological properties attributed to its secondary metabolites.
• In vitro studies aim to enhance these metabolites using elicitors and stresses, but molecular mechanisms require further investigation.
• Accurate gene expression analysis via RT-qPCR is crucial for understanding plant responses to treatments, yet relies on stable reference genes.

Purpose of the Study:

• To identify and validate stable reference genes for RT-qPCR in rosemary under various in vitro elicitor and stress conditions.
• To provide reliable normalization candidates for future gene expression studies in Salvia rosmarinus.
• To facilitate molecular research exploring rosemary's pharmacological potential.

Main Methods:

• Seven commonly used reference genes were evaluated for expression stability in rosemary under different in vitro treatments.
• RT-qPCR was employed to quantify reference gene expression.
• Expression stability was analyzed using five established algorithms: comparative ΔCt, BestKeeper, NormFinder, geNorm, and RefFinder.

Main Results:

• Expression stability varied significantly among the seven candidate reference genes under tested conditions.
• A combination of F1-ATPase, ATP synthase, and ACCase was identified as the most stable set for normalizing gene expression.
• The validated reference genes were confirmed using 4-coumarate-CoA ligase expression analysis.

Conclusions:

• F1-ATPase, ATP synthase, and ACCase are recommended as reliable reference genes for RT-qPCR in rosemary under in vitro conditions.
• This finding provides essential tools for accurate molecular studies in Salvia rosmarinus.
• The study also proposes reference genes for specific tissues, elicitors, and stress types, accelerating future research.