Identification and evaluation of reference genes for quantitative real-time PCR analysis in Polygonum cuspidatum...

Area of Science:

• Molecular Biology
• Plant Science
• Genomics

Background:

• Polygonum cuspidatum is a traditional medicinal plant with significant bioactive compounds.
• Accurate gene expression studies are crucial for understanding its health benefits and stress responses.
• Lack of validated reference genes hinders reliable gene expression analysis in P. cuspidatum.

Purpose of the Study:

• Identify and validate stable reference genes for quantitative real-time PCR (RT-qPCR) in Polygonum cuspidatum.
• Ensure accurate normalization of gene expression data across various experimental conditions.
• Facilitate advancements in molecular biology research for P. cuspidatum and related species.

Main Methods:

• Analyzed twelve candidate reference genes (nine common, three novel) in different tissues and under various abiotic stresses and hormone treatments.
• Assessed gene expression stability using multiple algorithms: △CT method, geNorm, NormFinder, BestKeeper, and RefFinder.
• Validated the suitability of identified reference genes by normalizing the expression of target genes (PcPAL, PcSTS, PcMYB4).

Main Results:

• NDUFA13 and EF-1γ were identified as the two most stable reference genes across all tested sample sets.
• These genes are also optimal for normalizing data from abiotic stresses and different tissues.
• NDUFA13 and SKD1 are recommended for hormone treatments; GAPDH and 60SrRNA showed stability under specific conditions.

Conclusions:

• NDUFA13 and EF-1γ are proposed as reliable reference genes for normalizing P. cuspidatum gene expression data.
• This study provides the first systematic validation of reference genes in P. cuspidatum.
• The findings will significantly enhance the reliability and accuracy of molecular studies in P. cuspidatum and related species.