Validation of seven housekeeping genes as reference ones for qRT-PCR normalization in Dendrobium catenatum

Abstract

Quantitative real-time polymerase chain reaction (qRT-PCR) has been applied to determine the expression levels of Dendrobium catenatum genes. However, so far, the suitable reference genes in the qRT-PCR analysis have not been established in this case. In this study, seven housekeeping genes of D. catenatum, i.e., those of actin 1 (ACT1), cyclophilin (CYP), eukaryotic initiation factor (eIF), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), α-tubulin (TUA), β-tubulin (TUB) and ubiquitin-conjugating enzyme (UBC), were selected as the candidate references for the qRT-PCR analysis of D. catenatum genes. Based on cDNA library, the cDNAs of the seven genes were cloned by reverse transcription (RT-PCR), and the transcription levels of these genes in different organs and tissues of D. catenatum were assessed by qRT-PCR; the expression stability of the genes was determined with BestKeeper, GeNorm and NormFinder programs, respectively. The open reading frames (ORFs) of the cDNAs of the seven genes were 1134, 522, 342, 1020, 1356, 1344 and 459 bp long, respectively. The corresponding amino acid sequences shared 87–100% identities with the prototype proteins registered in the GenBank, and held some specific conserved motifs required for the biological functions of these proteins. According to their expression stability, DcUBC, DcCYP and DcGAPDH would be preferentially selected as the reference genes for the qRT-PCR analysis of D. catenatum genes. Based on their expression levels, the most suitable reference ones were DcCYP, DcGAPDH and DcUBC in descending order.