Evaluation of candidate reference genes for qPCR in maize

Area of Science:

• Plant molecular biology
• Genomics and transcriptomics
• Biotechnology

Background:

• Quantitative real-time PCR (qPCR) is essential for measuring gene expression.
• Accurate normalization in qPCR relies on stable reference genes.
• No comprehensive stability assessments for maize reference genes were previously reported.

Purpose of the Study:

• To evaluate the expression stability of candidate reference genes in maize.
• To identify reliable reference genes for accurate qPCR data normalization in maize.
• To provide validated reference genes for future maize gene expression studies.

Main Methods:

• Analyzed expression patterns of 12 genes across 20 diverse maize samples (tissues, conditions).
• Utilized genorm(PLUS), NormFinder, and BestKeeper algorithms to assess gene expression stability.
• Compared stability of classical reference genes (ACT, TUB, 18S rRNA) with novel candidates.

Main Results:

• genorm(PLUS), NormFinder, and BestKeeper algorithms demonstrated comparable performance.
• Widely used maize reference genes (ACT, TUB, 18S rRNA) showed suboptimal expression stability.
• Five novel genes (CUL, FPGS, LUG, MEP, UBCP) exhibited the highest expression stability across all algorithms.

Conclusions:

• Established commonly used maize reference genes are not the most stable.
• Identified and validated five new reference genes (CUL, FPGS, LUG, MEP, UBCP) for maize qPCR.
• Provides essential tools for reliable gene expression analysis in maize research.