Selection of reference genes for RT-qPCR analysis in Coccinella septempunctata to assess un-intended effects of RNAi transgenic plants

Chunxiao Yang, Evan L. Preisser, Hongjun Zhang, Yong Liu, Liangying Dai, Huipeng Pan, Xuguo Zhou

Research output: Contribution to journalArticlepeer-review

32 Scopus citations


The development of genetically engineered plants that employ RNA interference (RNAi) to suppress invertebrate pests opens up new avenues for insect control. While this biotechnology shows tremendous promise, the potential for both nontarget and off-target impacts, which likely manifest via altered mRNA expression in the exposed organisms, remains a major concern. One powerful tool for the analysis of these un-intended effects is reverse transcriptase-quantitative polymerase chain reaction, a technique for quantifying gene expression using a suite of reference genes for normalization. The seven-spotted ladybeetle Coccinella septempunctata, a commonly used predator in both classical and augmentative biological controls, is a model surrogate species used in the environmental risk assessment (ERA) of plant incorporated protectants (PIPs). Here, we assessed the suitability of eight reference gene candidates for the normalization and analysis of C. septempunctata v-ATPase A gene expression under both biotic and abiotic conditions. Five computational tools with distinct algorisms, geNorm, Normfinder, BestKeeper, the ΔC t method, and RefFinder, were used to evaluate the stability of these candidates. As a result, unique sets of reference genes were recommended, respectively, for experiments involving different developmental stages, tissues, and ingested dsRNAs. By providing a foundation for standardized RT-qPCR analysis in C. septempunctata, our work improves the accuracy and replicability of the ERA of PIPs involving RNAi transgenic plants.

Original languageEnglish
Article number1672
JournalFrontiers in Plant Science
Issue numberNovember 2016
StatePublished - Nov 8 2016

Bibliographical note

Funding Information:
The authors are grateful to Hui Li for his assistance with the data analysis. This research was supported by a grant from USDA BRAG grant (3048108827), the National Natural Science Foundation of China (31501642), and a Special Fund for Agroscience Research in the Public Interest (201303028). The information reported in this paper (No. 16-08-026) is part of a project of the Kentucky Agricultural Experiment Station and is published with the approval of the Director.

Publisher Copyright:
© 2016 Yang, Preisser, Zhang, Liu, Dai, Pan and Zhou.


  • Coccinella septempunctata
  • Environmental risk assessment
  • Plant incorporated protectant
  • RNAi transgenic plants
  • RT-qPCR
  • Reference gene

ASJC Scopus subject areas

  • Plant Science


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