Selection and Validation of Appropriate Reference Genes for Quantitative Real-Time PCR Normalization in Staminate and Perfect Flowers of Andromonoecious Taihangia rupestris

Abstract

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used and powerful method for gene expression analysis due to its high sensitivity, specificity, and high throughput, and the accuracy of this approach depends on the stability of reference genes used for normalization. Taihangia rupestris Yu and Li (Rosaceae), an andromonoecious plant, produces both bisexual flowers and unisexual male flowers within the same individual. Using qRT-PCR technique, investigation of the gene expression profiling in staminate and perfect flowers would improve our understanding of the molecular mechanism in regulation of flower formation and sex differentiation in andromonoecious T. rupestris. To accurate normalize the gene expression level in Taihangia flower, 16 candidate reference genes, including 10 traditional housekeeping genes, and 6 newly stable genes, were selected based on transcriptome sequence data and previous studies. The expressions of these genes were assessed by qRT-PCR analysis in 51 samples, including 30 staminate and perfect flower samples across developmental stages and 21 different floral tissue samples from mature flowers. By using geNorm, NormFinder, BestKeeper, and comprehensive RefFinder algorithms, ADF3 combined with UFD1 were identified as the optimal reference genes for staminate flowers, while the combination of HIS3/ADF3 was the most accurate reference genes for perfect floral samples. For floral tissues, HIS3, UFD1, and TMP50 were the most suitable reference genes. Furthermore, two target genes, TruPI, and TruFBP24, involved in floral organ identity were selected to validate the most and least stable reference genes in staminate flowers, perfect flowers, and different floral tissues, indicating that the use of inappropriate reference genes for normalization will lead to the adverse results. The reference genes identified in this study will improve the accuracy of qRT-PCR quantification of target gene expression in andromonoecious T. rupestris flowers, and will facilitate the functional genomics studies on flower development and sex differentiation in the future.

Selection and Validation of Appropriate Reference Genes for Quantitative RT‐PCR Analysis in Rubia yunnanensis Diels Based on Transcriptome Data

“…Unexpectedly, a novel reference gene, hnRNP, vital for the translation of mRNA in the cytoplasm [50] also showed particularly excellent stability in all sample sets. Furthermore, increasing studies in various species [51][52][53] tended to apply multiple references rather than a single one considering the reliability and accuracy for the normalization of RT-qPCR data. In our study, the combination of two reference candidate genes has already satisfied the normalization under all the experimental conditions and various tissues ( Figure 5).…”

Reference genes forRT‐qPCRnormalisation in different tissues, developmental stages and stress conditions of amaranth

“…; Li et al . ); however, the identification of reference genes for the normalisation of RT‐qPCR has not been described in amaranth. This study evaluated genes to determine the most stable reference genes and analysed transcript stability of these genes in different tissues, developmental stages and under different types of stress in amaranth.…”

Selection and validation of reference genes for measuring gene expression in Toona ciliata under different experimental conditions by quantitative real-time PCR analysis

“…The rst three algorithms were used to evaluate the expression stability of candidate genes. Our results demonstrated that reference values and calculation methods used by the three algorithms were very different [40]. NormFinder calculates stability values based on intra-and intergroup differences [25], while geNorm compares a reference gene with all genes in a given sample to evaluate the best reference gene [24].…”