Selection and validation of reference genes for quantitative RT-PCR analysis in Castanea mollissima

Abstract

Quantitative RT-PCR (qRT-PCR) has been widely used in gene expression analysis because of its sensitivity, specificity, and reproducibility. Application of suitable reference genes to normalize qRT-PCR data is critical in analyzing PCR results. In this study, seven different tissues and organs (roots, stems, leaves, seed, flower buds, female flowers, male flowers) of Castanea mollissima were used as materials. The expression trends of six candidate internal reference genes (EF1α, TUA, TUB, UBQ, 18S rRNA, Actin) in chestnut were analyzed by RT-qPCR. The geNorm, NormFinder and BestKeeper software were used to analyze the expression stability of candidate internal reference genes. The selected internal reference gene was verified by the target gene CmSPL9. The results showed that Actin and EF1α had the most stability in different tissues and organs of C. mollissima. Actin, EF1α or Actin+EF1α can be used as reference test for C. mollissima internal reference genes.