Selection and validation of reference genes for quantitative RT-PCR analysis in peach fruit under different experimental conditions

Abstract

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a powerful tool for the detection and quantification of target gene expression levels. The accuracy of qRT-PCR data largely depends on the stability of the reference genes used for data normalization. Thus, the selection of suitable reference genes for qRT-PCR is necessary for accurate gene expression, especially under specific experimental conditions. In this study, stably expressed genes in peach fruit stored at various postharvest temperatures were screened based on RNA-seq data. Four newly identified stable genes and 12 traditional reference genes were selected as candidates. Their expression stability was examined in the peel and flesh of peach stored at five different temperatures (5°C, 15°C, 25°C, ambient temperature, and 35°C). Gene expression was further characterized in different plant tissues (root, stem, leaf, flower, and fruit) and fruit developmental stages. The overall performance of each candidate in all sample sets was also evaluated. As a result, the expression of PpeIF-1A was the most stable across the set of all samples, peel and flesh samples at different storage temperatures, and fruit developmental stages. PpGAPDH stability ranked highest in plant tissues. In general, PpeIF-1A and PpMUB6 were the most appropriate reference genes for all five experiments whereas PpRPS28, PpRPT-5A, and PpAKT3 were unsuitable control genes with variable expression patterns. The relative expression of the phytoene synthase gene PpPSY1 was assessed to confirm the utility of the selected reference genes in this study. Taken together, this study identified the reference genes suitable for the accurate and reliable normalization of peach gene expression data under different experimental conditions. The novel reference gene PpMUB6 exhibited stable expression compared to most of the traditional reference genes. These results provide guidance for selecting reliable internal control genes and will benefit future gene expression analysis in a wide variety of conditions in peach.