Reference gene selection for qRT-PCR in Brazilian-ginseng [Pfaffia glomerata (Spreng.) Pedersen] as affected by various abiotic factors

  • Diego Silva Batista
  • Viviane Santos Moreira
  • Sergio Heitor Sousa Felipe
  • Evandro Alexandre Fortini
  • Tatiane Dulcineia Silva
  • Kristhiano Chagas
  • Eliza Louback
  • Elisson Romanel
  • Marcio Gilberto Cardoso Costa
  • Wagner Campos OtoniEmail author
Original Article


The determination of the best reference gene is essential to improve and guarantee the accuracy of the qPCR technique. Thus, the objective of this work was to evaluate the normalization genes efficiency for qRT-PCR studies of Pfaffia glomerata, a species with marked medicinal and industrial interests due to the production of the phytoecdysteroid 20-hydroxyecdysone (20-E). We have selected four candidates as reference genes in P. glomerata: elongation factor- (EF-), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), spectrin-like (SPT) and α-tubulin (TUA), and tested their expression stabilities as affected by abiotic factors (salt stress, drought stress, irradiance, photoperiod and CO2 enrichment), using the following methods: NormFinder, geNorm, BestKeeper, ΔCt and RefFinder. Also, the Phantom gene, which belongs to the 20-E biosynthesis pathway, was targeted to validate the most stable reference gene for each abiotic factor. The GAPDH was the most stable gene under all assessed abiotic factors, as well as the most stable when data from all experiments were taken together, which was confirmed by all the software used. The use of more or less stable reference genes for normalization significantly changes the interpretation of the qPCR data, evidencing the importance of choosing the most appropriate housekeeping gene for each expression assay. Based on our results, we recommend GAPDH to be used for normalization of qPCR expression data in P. glomerata in diverse abiotic conditions. This work is the first report on the validation of reference genes in P. glomerata and will be fundamental for further gene expression studies in this important medicinal species.

Key message

GAPDH is the most stable gene to be used for the normalization in qPCR analyzes in [Pfaffia glomerata (Spreng.) Pedersen] under the different abiotic factors.


Abiotic conditions Brazilian ginseng Gene expression Quantitative real time Reference gene 



The authors thank the Brazilian sponsoring agencies, CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil), FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior), for financial support.

Authors contribution

DSB, SHSF, EAF, TDS, KC and EL conceived, designed and performed the experiments; DSB and VSM collected and analyzed the data; DSB, VSM, MGCC, and WCO contributed to the design and interpretation of the research and to the writing of the paper. All authors read and approved the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

11240_2019_1606_MOESM1_ESM.pdf (400 kb)
Supplementary material 1 (PDF 400 kb)


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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.PPG em Agricultura e AmbienteUniversidade Estadual do MaranhãoSão LuísBrazil
  2. 2.Instituto Federal da Bahia-Campus Euclides da Cunha–IFBAEuclides da CunhaBrazil
  3. 3.Laboratório de Cultura de Tecidos Vegetais (LCTII), Departamento de Biologia Vegetal/BIOAGROUniversidade Federal de ViçosaViçosaBrazil
  4. 4.Laboratório de Genômica de Plantas e Bioenergia (PGEMBL), Departamento de BiotecnologiaEEL/USPLorenaBrazil
  5. 5.Departamento de Ciências BiológicasUniversidade Estadual de Santa CruzIlhéusBrazil

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