Validation of candidate reference genes for gene expression normalization in Buchloe dactyloides using quantitative real-time RT-PCR

The proper selection of reference genes to normalize the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) results under special experimental conditions is crucial for quantifying gene expression. A systematic reference gene screening was performed using SYBR green qRT-PCR on 10 candidate genes in 27 biological samples of various photoperiodic treatments and different tissues in Buchloe dactyloides. Analysis by 3 different qRT-PCR methods (GeNorm, NormFinder, and BestKeeper) indicated that a combination of beta actin (β-ACT), dnaj protein gene (DNAJ) and protein phosphatase 2a (PP2A) were the most suitable reference genes across all samples examined. While the expression of the β-ACT and PP2A were the most stable genes in the long day (LD)-treated plant samples, glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) and β-ACT performed more stably in short day (SD) and constant dark (24D)-treated plant samples. β-ACT and PP2A displayed the most stable expression under constant light (24L) condition. β-ACT and DNAJ were the optimal reference genes in different tissues. Furthermore, examination of relative expression of a functional gene long hypocotyl 5 (HY5) in LD-treated RNA samples demonstrated additional reference genes contribute to accurate quantitative expression analysis. This study will help the selection of the most stable reference genes for accurate gene expression studies in B. dactyloides.