Selection of reliable reference genes for quantitative real-time PCR analysis in plum (Prunus salicina Lindl.) under different postharvest treatments

Abstract

The reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) technique has become one of the most widely used and reliable methods in gene expression studies. Successful application of qRT-PCR requires the accurate quantification of relative transcript levels, which strongly depends on the expression stability of the reference genes used as internal controls for data normalization. Plums (Prunus salicina Lindl.) are among the most numerous and commercial important fruit trees. In order to ensure the reliability of gene expression analyses using qRT-PCR in plum molecular biology research, 14 candidate reference genes were selected, and their relative expression levels were further measured by qRT-PCR using samples of plum peels obtained via different postharvest processes. Three statistical algorithms, geNorm, NormFinder, and BestKeeper, were employed to assess the expression stability of each candidate gene. A comprehensive evaluation was generated by the overall analysis approach, RefFinder to infer the final rankings. The results showed that CAC was the most stably expressed candidate reference gene across all experimental conditions. CAC and UNK under the room temperature treatment, CAC, ACT, and CLATH under the cold treatment, and CAC and ACT under all treatments were suitable for accurate gene expression quantification. In addition, relative gene expression patterns of the plant anthocyanin biosynthesis-related structural gene PsANS were evaluated using selected housekeeping genes as internal controls under two treatments to further confirm the usefulness of the selected reference genes. These results indicated that the selection of systematically validated reference genes for specific experimental conditions is necessary to avoid misinterpretation of qRT-PCR data and to obtain accurate and reliable gene expression results.