Evaluation of Candidate Reference Genes for Quantitative Gene Expression Analysis in Spodoptera exigua after Long-time Exposure to Cadmium

Anna Płachetka-Bożek,Maria Augustyniak

doi:10.1038/s41598-017-08630-6

Abstract

Studies on the transcriptional control of gene expression play an important role in many areas of biology. Reference genes, which are often referred to as housekeeping genes, such as GAPDH, G3PDH, EF2, RpL7A, RpL10, TUBα and Actin, have traditionally been assumed to be stably expressed in all conditions, and they are frequently used to normalize mRNA levels between different samples in qPCR analysis. However, it is known that the expression of these genes is influenced by numerous factors, such as experimental conditions. The difference in gene expression underlies a range of biological processes, including development, reproduction and behavior. The aim of this study was to show the problems associated with using reference genes in the qPCR technique, in a study on inbred strains of Spodoptera exigua selected toward cadmium resistance. We present and discuss our results and observations, and give some recommendations concerning the use and limitations of housekeeping genes as internal standards, especially in research on insects. Our results suggest that holometabolism and poikilothermia, as well as time since metamorphosis and the level of exposure to the selective factor (cadmium in this case), have a significant effect on the expression of reference genes.

Highlights

The perfect reference genes should be expressed in abundance and have minimal innate variability[2]
The mRNA level of housekeeping genes can depend on numerous factors, such as the stage of an organism’s development, cell cycle, experimental conditions[5], time of day when the material was collected, etc
In the last decade, when performing various mRNA quantification experiments, most scientists observed that expression levels of housekeeping genes that are used as internal standards can fluctuate[5]

Summary

Introduction

The perfect reference genes should be expressed in abundance and have minimal innate variability[2]. Verma and Shapiro (2006) discovered and described sex-dependent expression of several genes that are commonly used as reference genes in real-time PCR7. This team reported constitutive sexual dimorphism of hepatic mRNA level of seven commonly measured housekeeping genes such as tubulin, glyceraldehyde 3-phosphate dehydrogenase, β-actin and ribosomal protein 18S in rat liver. This is probably connected with varying hormonal regulation, the particular hormone(s) involved have not been identified[7]. It was possible to assess the usability of a wide array of reference genes in research done on insects after complete metamorphosis

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