Selection and validation of reference genes for quantitative gene expression analyses in various tissues and seeds at different developmental stages in Bixa orellana L.
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Bixa orellana L., popularly known as annatto, produces several secondary metabolites of pharmaceutical and industrial interest, including bixin, whose molecular basis of biosynthesis remain to be determined. Gene expression analysis by quantitative real-time PCR (qPCR) is an important tool to advance such knowledge. However, correct interpretation of qPCR data requires the use of suitable reference genes in order to reduce experimental variations. In the present study, we have selected four different candidates for reference genes in B. orellana, coding for 40S ribosomal protein S9 (RPS9), histone H4 (H4), 60S ribosomal protein L38 (RPL38) and 18S ribosomal RNA (18SrRNA). Their expression stabilities in different tissues (e.g. flower buds, flowers, leaves and seeds at different developmental stages) were analyzed using five statistical tools (NormFinder, geNorm, BestKeeper, ΔCt method and RefFinder). The results indicated that RPL38 is the most stable gene in different tissues and stages of seed development and 18SrRNA is the most unstable among the analyzed genes. In order to validate the candidate reference genes, we have analyzed the relative expression of a target gene coding for carotenoid cleavage dioxygenase 1 (CCD1) using the stable RPL38 and the least stable gene, 18SrRNA, for normalization of the qPCR data. The results demonstrated significant differences in the interpretation of the CCD1 gene expression data, depending on the reference gene used, reinforcing the importance of the correct selection of reference genes for normalization.
KeywordsNormalizer genes Endogenous genes Quantitative real time Stability Gene expression
This work was supported by research grants from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brasília, Brazil), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasília, Brazil) and UESC (Universidade Estadual de Santa Cruz, Ilhéus, Bahia, Brazil). We gratefully acknowledge the Ph.D. scholarship provided by CAPES Foundation to VSM.
VSM conducted the experiments. VSM, VLFS, RJSS and AOS analyzed the data. VSM drafted the manuscript. VLFS, WCO and MGCC supported the project and designed the experiments. VLFS and MGCC revised the manuscript. All authors read and approved the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
Research involving human participants and/or animals
The authors declare that the present study does not involve any human participants and/or animals.
The authors declare that the present study does not involve any informed consent.
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