Selection of appropriate reference genes for RT-qPCR analysis in Propylea japonica (Coleoptera: Coccinellidae)

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique commonly used in molecular biology to analyze RNA expression. The selection of suitable reference genes for data normalization is a precondition for credible measurements of gene expression levels using RT-qPCR. Propylea japonica is one of the most common pests of many crop systems throughout East Asia, and has often been used in the testing of non-target impacts during environmental risk assessments of genetically engineered plants. The present study assessed the suitability of nine frequently used reference genes for comparisons of P . japonica gene expression. Expression stability was compared across developmental stages, sex, a range of tissues, and following exposure to different temperatures. Data were analyzed using RefFinder , which integrated the results obtained using NormFinder , geNorm , BestKeeper , and the ΔCt method. This led to the identification of unique sets of reference genes for each experimental condition: ribosomal protein S18 ( RPS18 ) and elongation factor 1 α ( EF1A ) for developmental stage comparisons, RPS18 and EF1A for sex comparisons, EF1A and ribosomal protein L4 for tissue comparisons, and RPS18 and EF1A for analyses of temperature-mediated effects. These reference genes will help to enhance the accuracy of RT-qPCR analyses of P . japonica gene expression. This work represents an initial move towards building a standardized system for RT-qPCR analysis of P . japonica , providing a basis for the ecological risk assessment of RNAi-based insect control products.