Selection and validation of reference genes for quantitative gene expression analyses in various tissues and seeds at different developmental stages in Bixa orellana L.

Autor: Moreira VS; 1Departamento de Ciências Biológicas, Universidade Estadual de Santa Cruz, Ilhéus, Bahia 45662-900 Brazil., Soares VLF; 1Departamento de Ciências Biológicas, Universidade Estadual de Santa Cruz, Ilhéus, Bahia 45662-900 Brazil., Silva RJS; 1Departamento de Ciências Biológicas, Universidade Estadual de Santa Cruz, Ilhéus, Bahia 45662-900 Brazil., Sousa AO; 2Departamento de Tecnologia e Ciências Sociais, Universidade do Estado da Bahia, Juazeiro, Bahia 48905-680 Brazil., Otoni WC; 3Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Viçosa, Minas Gerais 36570-900 Brazil., Costa MGC; 1Departamento de Ciências Biológicas, Universidade Estadual de Santa Cruz, Ilhéus, Bahia 45662-900 Brazil.
Jazyk: angličtina
Zdroj: Physiology and molecular biology of plants : an international journal of functional plant biology [Physiol Mol Biol Plants] 2018 May; Vol. 24 (3), pp. 369-378. Date of Electronic Publication: 2018 Apr 03.
DOI: 10.1007/s12298-018-0528-1
Abstrakt: 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.
Competing Interests: Compliance with ethical standardsThe authors declare that they have no competing interests.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.
Databáze: MEDLINE
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