Selection of Reference Genes for the Normalization of RT-qPCR Data in Gene Expression Studies in Insects: A Systematic Review

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for quantifying expression levels of targeted genes during various biological processes in numerous areas of clinical and biological research. Selection of appropriate reference genes for RT-qPCR normalization is an elementary prerequisite for reliable measurements of gene expression levels. Here, by analyzing datasets published between 2008 and 2017, we summarized the current trends in reference gene selection for insect gene expression studies that employed the most widely used SYBR Green method for RT-qPCR normalization. We curated 90 representative papers, mainly published in 2013-2017, in which a total of 78 insect species were investigated in 100 experiments. Furthermore, top five journals, top 10 frequently used reference genes, and top 10 experimental factors have been determined. The relationships between the numbers of the reference genes, experimental factors, analysis tools on the one hand and publication date (year) on the other hand was investigated by linear regression. We found that the more recently the paper was published, the more experimental factors it tended to explore, and more analysis tools it used. However, linear regression analysis did not reveal a significant correlation between the number of reference genes and the study publication date. Taken together, this meta-analysis will be of great help to researchers that plan gene expression studies in insects, especially the non-model ones, as it provides a summary of appropriate reference genes for expression studies, considers the optimal number of reference genes, and reviews the average number of experimental factors and analysis tools per study.