Selection and validation of stable reference genes in guava (Psidium guajava L.) for reliable and consistent gene expression analysis

Selection and validation of stable reference genes in guava ( Psidium guajava L.) for reliable and consistent gene expression analysis Ideal stably expressing housekeeping gene to be used as internal control needs to be optimized for efficient gene expression analysis for accurate mRNA quantitation. In pursuit of identifying suitable reference genes for gene expression analysis in guava, a systematic study was conducted using different tissues of guava cv. Allahabad Safeda examining 10 housekeeping genes such as Actin ( PgACT ), Elongation factor 1G ( PgEF1G ), Elongation factor 2 ( PgEF2 ), Tubulin ( PgTUB1 ), Elongation factor 1 α ( PgEF1a ), Monensin sensitivity1 ( PgMON1 ), Histone H3 ( PgH3 ), RNA-binding protein 47 ( PgRBP47 ), Polyubiquitin_X1 ( PgPOLX1 ), and Polyubiquitin_X2 ( PgPOLX2 ). qRT-PCR analysis showed amplification efficiencies ranging from 74.4% to 124.9%, with correlation coefficients exceeding 0.98. The stability of these genes’ expression was evaluated using six methods: GeNorm, NormFinder, BestKeeper, RefFinder, comparative delta-Ct, and Stability index in which different methods identified PgRBP47 as least stable and indicated its unsuitability as a reference gene but showed variations in the ranking of the genes for gene stability. Comparison of all the methods and accounting for the top 3 ranks of gene stability, three genes i.e., PgTUB1 , PgEF1a , and PgEF2 were identified as more stable housekeeping genes across different tissues of guava and could be considered as ideal reference genes for normalization in gene expression studies of guava. How to cite: Kumar S, Muthukumar M, Bajpai A, et al. Selection and validation of stable reference genes in guava ( Psidium guajava L.) for reliable and consistent gene expression analysis. Electron J Biotechnol 2025;75. https://doi.org/10.1016/j.ejbt.2025.01.006 .