Identification and validation of housekeeping genes in brains of the desert locust Schistocerca gregaria under different developmental conditions

Matthias B Van Hiel,Roger Huybrechts,Liesbet Temmerman,Pieter Van Wielendaele,Kristel Vuerinckx,Jozef Broeck,Sofie Van Soest,Gert Simonet

doi:10.1186/1471-2199-10-56

Abstract

BackgroundTo obtain reliable quantitative RT-PCR data, normalization relative to stable housekeeping genes is required. However, in practice, expression levels of 'typical' housekeeping genes have been found to vary between tissues and under different experimental conditions. To date, validation studies of reference genes in insects are extremely rare and have never been performed in locusts. In this study, putative housekeeping genes were identified in the desert locust, Schistocerca gregaria and two different software programs (geNorm and Normfinder) were applied to assess the stability of thesegenes.ResultsWe have identified seven orthologs of commonly used housekeeping genes in the desert locust. The selected genes were the orthologs of actin, EF1a, GAPDH, RP49, TubA1, Ubi, and CG13220. By employing real time RT-PCR we have analysed the expression of these housekeeping genes in brain tissue of fifth instar nymphs and adults. In the brain of fifth instar nymphs geNorm indicated Sg-EF1a, Sg-GAPDH and Sg-RP49 as most stable genes, while Normfinder ranked Sg-RP49, Sg-EF1a and Sg-ACT as most suitable candidates for normalization. The best normalization candidates for gene expression studies in the brains of adult locusts were Sg-EF1a, Sg-GAPDH and Sg-Ubi according to geNorm, while Normfinder determined Sg-GAPDH, Sg-Ubi and Sg-ACT as the most stable housekeeping genes.ConclusionTo perform transcript profiling studies on brains of the desert locust, the use of Sg-RP49, Sg-EF1a and Sg-ACT as reference genes is proposed for studies of fifth instar nymphs. In experiments with adult brains, however, the most preferred reference genes were Sg-GAPDH, Sg-Ubi and Sg-EF1a. These data will facilitate transcript profiling studies in desert locusts and provide a good starting point for the initial selection of genes for validation studies in other insects.

Highlights

To obtain reliable quantitative RT-PCR data, normalization relative to stable housekeeping genes is required
Using the quantitative real-time RT-PCR technique the expression levels of a gene can be investigated in different cells, tissues and organisms and in different conditions during development or over a preferred period of time. qPCR is widely used to verify microarray datasets or the knockdown of a gene in RNA interference experiments and is of great value in disease diagnostics [1,2,3]
In this study we identified and examined seven housekeeping genes (HKGs) in brain tissues of desert locusts (Schistocerca gregaria) during the last molt and the reproductive cycle

Summary

Introduction

To obtain reliable quantitative RT-PCR data, normalization relative to stable housekeeping genes is required. The analysis of qPCR data requires normalization relative to an active reference or endogenous control, which compensates for differences in sample preparation, cDNA and DNA synthesis and in the amount of the starting material Such an internal control gene ideally has an equal transcript level in all cells at every developmental stage and is unaffected by experimental conditions. It was shown that transcript levels, normalized to a single HKG, can differ more than 20-fold from the actual expression [5] To circumvent this problem tests to validate the stability of HKGs and the use of multiple genes are supported. To this end, different software programs were developed to make a selection of housekeeping genes that are most suited for normalization [4,6,7]

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