Evaluation of new reference genes in papaya for accurate transcript normalization under different experimental...

Area of Science:

• Plant molecular biology
• Gene expression analysis
• Biotechnology

Background:

• Real-time reverse transcription PCR (RT-qPCR) is essential for quantifying gene expression.
• Accurate normalization using stable reference genes is critical for RT-qPCR reliability.
• No universal reference genes exist, necessitating validation for specific experimental conditions, especially in papaya.

Purpose of the Study:

• To systematically evaluate the expression stability of candidate reference genes in papaya under diverse experimental conditions.
• To identify the most suitable reference gene(s) for accurate transcript normalization in papaya.
• To provide guidelines for selecting reference genes in papaya gene expression studies.

Main Methods:

• Selected 21 candidate reference genes for evaluation.
• Analyzed 246 papaya fruit samples across 13 distinct experimental conditions.
• Utilized geNorm, NormFinder, and RefFinder algorithms to assess gene expression stability.

Main Results:

• Reference gene expression stability varied significantly across different experimental conditions.
• Eukaryotic initiation factor 4A (EIF), TATA binding protein 1 (TBP1), and TATA binding protein 2 (TBP2) demonstrated good stability under most conditions.
• Commonly used genes like Actin 2 (ACTIN), 18S ribosomal RNA (18S rRNA), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were often unsuitable.

Conclusions:

• Reference gene selection must be condition-specific for accurate RT-qPCR in papaya.
• EIF, TBP1, and TBP2 are recommended as reliable reference genes for papaya.
• This study offers the first comprehensive analysis for superior reference gene selection in papaya.