Selection of suitable reference genes for reverse transcription quantitative real-time PCR studies on different experimental systems from carrot (Daucus carota L.)

Abstract

Reverse transcription quantitative real time polymerase chain reaction (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization through the use of suitable reference genes. This study aimed selecting robust and reliable reference genes which are constitutively and equally expressed for accurate RT-qPCR normalization analysis in two different experimental systems with carrot. A systematic comparison of 12 selected candidate genes for carrot is presented. These included seven genes commonly used as reference gene: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), 18S ribosomal RNA (18S rRNA), ubiquitin (UBQ), alpha-tubulin (TUA), beta-tubulin (β-TUB) and elongation factor-1alpha (EF-1A). Additionally, other five genes were here presented as potential candidates: other ribosomal RNA (25S and 5.8S rRNA) and ribosomal protein L2 encoding gene (rpL2), the transcriptional initiation factor (TIF1), and the heat shock protein 70 (hsp70). Expression stability was evaluated in: (i) in vivo growth experiment based on carrot tap root secondary growth carried out in growth chambers and (ii) realization phase of somatic embryogenesis. During carrot tap root secondary growth, two reference genes GAPDH and 5.8S rRNA, were stably expressed. In the somatic embryogenesis realization experiment, two ribosomal RNAs were selected as reference genes, the 5.8S and the 25S rRNA. Additionally, the expression profile of the mitochondrial alternative oxidase gene DcAOX1 was conducted in the in vivo growth experiment, to show the impact of reference genes selection. Taken together, our results provide a systematic analysis for the selection of superior reference genes for accurate transcript normalization in carrot, under different experimental conditions. We reinforce the idea that the validation of reference genes for the conditions under study is essential, as emphasized by the expression analysis of DcAOX1 as target gene. These results show that a previous selection of reference genes for each experimental system is crucial to achieve accurate and reliable RT-qPCR gene expression data, avoiding low precision or misleading results.