Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable, rapid, and reproducible technique for measuring and evaluating changes in gene expression. To facilitate gene expression studies and obtain more accurate RT-qPCR data, normalization relative to stable reference genes is required. In this study, expression profiles of seven candidate reference genes, including β-actin (Actin), elongation factor 1 α (EF1A), glyceralde hyde-3-phosphate dehydro-genase (GAPDH), cyclophilins A (CypA), vacuolar-type H+-ATPase (ATPase), 28S ribosomal RNA (28S), and 18S ribosomal RNA (18S) from Hippodamia convergens were investigated. H. convergens is an abundant predatory species in the New World, and has been widely used as a biological control agent against sap-sucking insect pests, primarily aphids. A total of four analytical methods, geNorm, Normfinder, BestKeeper, and the ΔCt method, were employed to evaluate the performance of these seven genes as endogenous controls under diverse experimental conditions. Additionally, RefFinder, a comprehensive evaluation platform integrating the four above mentioned algorithms, ranked the overall stability of these candidate genes. A suite of reference genes were specifically recommended for each experimental condition. Among them, 28S, EF1A, and CypA were the best reference genes across different development stages; GAPDH, 28S, and CypA were most stable in different tissues. GAPDH and CypA were most stable in female and male adults and photoperiod conditions, 28S and EF1A were most stable under a range of temperatures, Actin and CypA were most stable under dietary RNAi condition. This work establishes a standardized RT-qPCR analysis in H. convergens. Additionally, this study lays a foundation for functional genomics research in H. convergens and sheds light on the ecological risk assessment of RNAi-based biopesticides on this non-target biological control agent.

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Published in PLOS One, v. 10, no. 4, article e0125868, p. 1-15.

© 2015 Pan et al.

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

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This research was supported by a start-up fund from the University of Kentucky to XGZ, a grant from USDA BRAG grant (Award Agreement No.: 3048108827) to XGZ and BDS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

S1_Fig.tif (1958 kB)
S1 Fig. Melting curve of the seven candidate reference genes.

S1_Table.docx (11 kB)
S1 Table. Degenerate primers used for RT-qPCR.

S2_Table.docx (14 kB)
S2 Table. The sequencing results of these five genes including Actin, EF1A, GAPDH, CypA, ATPase using the degenerate primers.

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