A Comprehensive Selection of Reference Genes for RT-qPCR Analysis in a Predatory Lady Beetle, Hippodamia convergens (Coleoptera: Coccinellidae)

• Published in: PloS one 2015-04, Vol.10 (4), p.e0125868-e0125868
• Main Authors: Pan, Huipeng, Yang, Xiaowei, Siegfried, Blair D, Zhou, Xuguo
• Format: Article
• Language: English
• Subjects: Actin; Acyrthosiphon pisum; Adenosine triphosphatase; Adults; Algorithms; Analytical methods; Animals; Aphidoidea; Aphids; ATPases; Bactrocera dorsalis; Beetles; Biological control; Biological pest control; Coccinellidae; Coleoptera; Coleoptera - genetics; Convergence; Developmental stages; Diet; DNA - genetics; DNA polymerases; Ecological risk assessment; Elongation; Female; Gene expression; Genes; Genes, Insect - genetics; Genomics; Glyceraldehyde-3-phosphate dehydrogenase; H+-transporting ATPase; Hemiptera; Hippodamia convergens; Insect pests; Insects; Ladybugs; Lepidoptera; Male; Muscle proteins; Peptidylprolyl isomerase; Pesticides; Pests; Phosphates; Polymerase chain reaction; Reverse Transcriptase Polymerase Chain Reaction - methods; Reverse Transcriptase Polymerase Chain Reaction - standards; Ribonucleic acid; Risk assessment; RNA; RNA-directed DNA polymerase; RNA-mediated interference; rRNA 18S; rRNA 28S; Stem cells; Temperature range; Tissues
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0125868

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.