TY - JOUR
T1 - Selection of reference genes for quantitative PCR analysis in Citrus aurantifolia during phytoplasma infection
AU - Alves, Murilo S.
AU - Al-Sadi, Abdullah M.
AU - Carvalho, Claudine M.
N1 - Funding Information:
Acknowledgments This work was funded by Vale S.A. MSA was a Vale Technological Institute postdoctoral fellow. CMC is the recepient of a CNPq research productivity fellowship. We thank Sam Elliot (Dep. de Entomologia, UFV) for critically reviewing the English of the manuscript.
Funding Information:
This work was funded by Vale S.A. MSA was a Vale Technological Institute postdoctoral fellow. CMC is the recepient of a CNPq research productivity fellowship. We thank Sam Elliot (Dep. de Entomologia, UFV) for critically reviewing the English of the manuscript. The authors declare that they have no conflict of interest.
Publisher Copyright:
© 2018, Sociedade Brasileira de Fitopatologia.
PY - 2018/10/1
Y1 - 2018/10/1
N2 - 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.
AB - 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.
KW - Citrus
KW - Normalization
KW - Phytoplasma
KW - Reference gene
KW - qPCR
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U2 - 10.1007/s40858-018-0224-2
DO - 10.1007/s40858-018-0224-2
M3 - Article
AN - SCOPUS:85054163267
SN - 1982-5676
VL - 43
SP - 402
EP - 412
JO - Tropical Plant Pathology
JF - Tropical Plant Pathology
IS - 5
ER -