Validation of endogenous reference genes in Buglossoides arvensis for normalizing RT-qPCR-based gene expression data.

Area of Science:

• Plant molecular biology
• Gene expression analysis
• Nutraceutical crop development

Background:

• Accurate gene expression studies rely on stable reference genes (RGs) for normalization.
• Buglossoides arvensis (Ahiflower™) is a valuable plant for its high omega-3 poly-unsaturated fatty acid (PUFA) content.
• Validation of RGs is crucial for non-model plants like B. arvensis.

Purpose of the Study:

• To identify and validate stable reference genes for quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) in B. arvensis.
• To ensure reliable gene expression data for research and commercial applications of Ahiflower™.
• To establish a foundation for future molecular studies in B. arvensis.

Main Methods:

• Ten candidate reference genes were selected and isolated from B. arvensis.
• TaqMan™ compliant primers and probes were designed for RT-qPCR.
• Gene transcript abundance was analyzed across various tissues and growth conditions using geNorm and NormFinder algorithms.

Main Results:

• Candidate reference gene expression varied across tissues and growth regimes.
• geNorm and NormFinder analyses indicated differing stability levels for the tested RGs.
• Combinatorial analysis identified CAC as the most stable RG and α-actin as the least stable RG.

Conclusions:

• This study provides the first validated reference genes for B. arvensis.
• CAC is recommended as a stable reference gene for RT-qPCR in B. arvensis.
• Validated RGs will enhance the reliability of gene expression studies in this important nutraceutical crop.