Selection of reference genes for quantitative PCR analysis in Citrus aurantifolia during phytoplasma infection

Murilo S. Alves, Abdullah M. Al-Sadi, Claudine M. Carvalho*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)


Quantifying gene expression is essential in most functional genomics experiments. For quantitative PCR (qPCR) assays, reproducible results are dependent on the correct choice of the reference genes for data normalization. To date, screenings for candidate reference genes suitable for expression studies on plant-phytoplasma interactions in plants have not been reported. In the present study, we analyzed the expression patterns of 14 genes in midrib samples of C. aurantifolia plants infected with a ‘Candidatus Phytoplasma aurantifolia’ strain. Using GeNormPlus, NormFinder and BestKeeper algorithms, as well as testing relative expression by REST2009 software, the expression stability of several “classical” reference genes, such as GAPDH, CYCLOPHILIN and 18S rRNA, and of newly identified candidates, was assessed. Our results showed similar performance among GeNormPlus, NormFinder and BestKeeper in evaluating the suitability of reference genes, with few differences among the top five genes. Furthermore, our data showed that some of the widely used reference genes for relative expression normalization in plants, including citrus lineages, were not the most stably expressed transcripts. In conclusion, we provide a list of validated reference genes and their relative primer sequences, usable to conduct reliable qPCR experiments in C. aurantifolia during phytoplasma infection.

Original languageEnglish
Pages (from-to)402-412
Number of pages11
JournalTropical Plant Pathology
Issue number5
Publication statusPublished - Oct 1 2018


  • Citrus
  • Normalization
  • Phytoplasma
  • Reference gene
  • qPCR

ASJC Scopus subject areas

  • Agronomy and Crop Science
  • Plant Science


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