Transcriptome-based validation of proper reference genes for reverse trascription quantitative PCR analysis of Sinocalycanthus chinensis

Abstract

Reverse transcription quantitative PCR is a widely used method to detect gene expressions. To obtain accurate expression results, the selection of proper reference genes is important and necessary. However, related works concerning reference gene selection have not been carried on many plant species, especially endangered ones. The aim of the present study was to select dependable reference genes for expression normalization of an endangered plant species with medicinal and ornamental value: Sinocalycanthus chinensis (Calycanthaceae). Nine reference genes with stable expressions were chosen for further analysis according to transcriptomic sequencing data from S. chinensis. The expression stability of these candidate genes in inner and outer petals at different floral developmental stages and in many different tissues was then analyzed with the geNorm and NormFinder software. The reference genes were evaluated by normalizing the expression of the anthocyanidin synthase gene in the outer and inner petals at different floral developmental stages to further verify the expression stability of these genes. Elongation factor 1-alpha (ScEF) and 50S ribosomal protein L27 were found to be the two most stable genes in the overall ranking of all the samples and different tissues. Furthermore, ScEF and protein phosphatase 2A were stably expressed in all petal samples. Moreover, among the nine candidate reference genes, phosphoglycerate kinase performed poorly in all sample sets. Our results will help to obtain reliable expression data in molecular studies of S. chinensis.

Screening and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR under PEG, NaCl and ZnSO4 Treatments in Broussonetia papyrifera

“…The screening of the internal reference genes in combination with the plant transcriptome database is one of the most effective methods for the research in nonmodel plants [31]. It has been applied in various plants such as Malpighia emarginata [32], Sinocalycanthus chinensisb [33], and Oryza sativab [34]. Therefore, through the transcriptome database, this study screened a batch of stable candidate internal reference genes, NADH, L13, EIF3, HIS, Actin, PP2A, DOUB, UBE2, UBC, PTB, rRNA, GAPDH, HSP, RPL8, and TUA.…”

Selection of Suitable Reference Genes Based on Transcriptomic Data in Ginkgo biloba under Different Experimental Conditions

“…To better normalize the expression data, selection of reference genes based on transcriptomic data has been performed in many plant species, such as Eucalyptus globulus [68], Oxytropis ochrocephala [42], Arabidopsis pumila [69], Sinocalycanthus chinensis [70], and Polygonum cuspidatum [71]. In our study, the thirteen candidate reference genes were chosen based on transcriptome sequencing data.…”