Identification and evaluation of reference genes for reliable normalization of real-time quantitative PCR data in acerola fruit, leaf, and flower

  • Clesivan Pereira dos Santos
  • Kátia Daniella da Cruz Saraiva
  • Mathias Coelho Batista
  • Thais Andrade Germano
  • José Hélio CostaEmail author
Original Article


Understanding into acerola (Malpighia emarginata) molecular and biochemical bases is still obscure, despite it is one of the most important natural source of vitamin C for humans. Recently, our research group published the first data on acerola transcriptome generating valuable information to identify reference genes for RT-qPCR in this species. Hence, this study aimed to identify the most stably expressed genes based on acerola transcriptome data, and further to evaluate the suitability of F-box, U3, Merad50-ATPase, TGD4, NOB1, PA-RNA, RCC1, RBL and PGAL candidates for accurate gene expression normalization in leaf, flower and fruit at 12, 16 and 20 days after anthesis using RT-qPCR analysis. Three algorithms, geNorm, NormFinder, and BestKeeper confirmed the expression stability of all nine candidate reference genes, whereas RefFinder consensually summarized a comprehensive gene ranking. Based on geNorm, the combination of the most stable reference genes RBL and U3 for leaf/flower group, TGD4, F-box and PGAL (fruit developmental stages or fruit/leaf), RCC1, PGAL and RBL (fruit/flower) and RCC1, RBL, TGD4 and PGAL (total samples) were required for accurate normalization. Moreover, the use of these reference genes to assess the expression profile of GMP1 and NAT3 genes confirmed the reliability of ranking and defined the best combination of genes recommended by geNorm and RefFinder. This work will benefit further RT-qPCR studies in these acerola organs by offering a foundation for accurate normalization of gene expression profiling.


Acerola Reference genes RT-qPCR Expression stability Validation 


Author contributions

CPS, KDCS, and JHC conceived and designed research. CPS, KDCS and, TAG performed the experiments. CPS, KDCS, and MCB analyzed data. CPS wrote the manuscript. All authors read, commented on, and approved the manuscript.


This research was supported by CNPq, CAPES, and FUNCAP. CPS acknowledge doctoral grants from CNPq. JHC is grateful to CNPq for the Researcher fellowship (CNPq Grant 309795/2017-6).

Compliance with ethical standards

Conflict of interest

The authors have no conflict of interest to declare.

Supplementary material

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Supplementary material 1 (DOC 5076 kb)
11033_2019_5187_MOESM2_ESM.xls (100 kb)
Supplementary material 2 (XLS 100 kb)
11033_2019_5187_MOESM3_ESM.doc (42 kb)
Supplementary material 3 (DOC 41 kb)


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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Functional Genomics and Bioinformatics, Department of Biochemistry and Molecular BiologyFederal University of CearáFortalezaBrazil
  2. 2.Federal Institute of Education, Science and Technology of ParaíbaPrincesa IsabelBrazil

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