Housekeeping in Tephritid insects: the best gene choice for expression analyses in the medfly and the olive fly

Efthimia Sagri,Yiannis C Bassiakos,Panagiota Koskinioti,Konstantina T Tsoumani,Maria-Eleni Gregoriou,Kostas D Mathiopoulos

doi:10.1038/srep45634

Efthimia Sagri, Yiannis C Bassiakos + Show 4 more

Open Access

https://doi.org/10.1038/srep45634

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Abstract

Real-time quantitative-PCR has been a priceless tool for gene expression analyses. The reaction, however, needs proper normalization with the use of housekeeping genes (HKGs), whose expression remains stable throughout the experimental conditions. Often, the combination of several genes is required for accurate normalization. Most importantly, there are no universal HKGs which can be used since their expression varies among different organisms, tissues or experimental conditions. In the present study, nine common HKGs (RPL19, tbp, ubx, GAPDH, α-TUB, β-TUB, 14-3-3zeta, RPE and actin3) are evaluated in thirteen different body parts, developmental stages and reproductive and olfactory tissues of two insects of agricultural importance, the medfly and the olive fly. Three software programs based on different algorithms were used (geNorm, NormFinder and BestKeeper) and gave different ranking of HKG stabilities. This confirms once again that the stability of common HKGs should not be taken for granted and demonstrates the caution that is needed in the choice of the appropriate HKGs. Finally, by estimating the average of a standard score of the stability values resulted by the three programs we were able to provide a useful consensus key for the choice of the best HKG combination in various tissues of the two insects.

Highlights

The study of any gene inevitably goes through detailed and thorough scrutiny of its expression profile in various tissues and under different conditions
Three available software programs were used for the analysis and, since each program is based on a different algorithm, an effort was put to generate a consensus of the three programs
Several times in the recent years it has been documented that the choice of the right reference gene/s for the standardization of RT-qPCRs is of paramount importance and the possible use of the inappropriate housekeeping gene (HKG) can lead to incorrect results[11,12,13,14]

Summary

Introduction

The study of any gene inevitably goes through detailed and thorough scrutiny of its expression profile in various tissues and under different conditions. Many articles have been published on the identification and selection of the best reference gene in specific tissues and under different conditions. Normalization of RT-qPCR is at best performed using a housekeeping gene (HKG) that demonstrates stable expression in microarray or RNAseq results[26,27]. This strategy may seem biologically reasonable, but there is a potential technical artifact considering that microarrays, RNAseq and RT-qPCR constitute quite different methods, with different limitations, requiring different standardization each. Neither the same genes nor the same tissues and conditions are studied, a fact that makes any effort to compare results practically impossible. We analyzed three developmental stages (egg, larva, pupa) and the three sections of the insect body (head, thorax, abdomen), as they are often convenient controls for comparison with other tissues

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