Abstract
BackgroundQuantitative RT-PCR (q-RT-PCR) is a powerful tool that allows for the large scale analysis of small changes in gene expression. Accurate and reliable results depend on the use of stable reference genes for normalization. However, the expression of some widely used housekeeping genes can vary under different experimental setups. To our knowledge, no validation studies have been reported for reference genes in cockroaches. The aim of the current study is the identification and validation of a set of eight housekeeping genes during the first gonadotrophic cycle of the cockroach, Diploptera punctata. This study made use of two different algorithms (geNorm and Normfinder) to evaluate the stability of gene expression.ResultsCandidate housekeeping genes were sequenced: β-actin (Actin), elongation factor 1 alpha (EF1a), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), armadillo (Arm), ribosomal protein L32 (RpL32), succinate dehydrogenase (SDHa), annexin IX (AnnIX) and α-tubulin (Tub). The expression of these eight genes was analyzed in corpora allata (CA) and ovaries of adult female D. punctata. Both geNorm, as well as Normfinder characterized SDHa, EF1a and Arm as being the most stably expressed in the corpora allata. In the ovary, the geNorm calculation showed Tub, EF1a and RpL32 to be most stable, whereas Normfinder identified Tub, EF1a and Arm as the best. In ovary, the least stable gene was Actin, challenging its usefulness in normalization. As a proof of principle, the expression of follicle cell protein 3c and CYP15A1 was monitored during the first gonadotrophic cycle.ConclusionArm and EF1a form the most stably expressed combination of two reference genes out of the eight candidates that were tested in the corpora allata. Our results show that the combined use of Tub, EF1a and RpL32 ensures an accurate normalization of gene expression levels in ovary of D. punctata. Our study has indicated that neither Actin nor AnnIX should be used for normalization of transcript levels when studying the first gonadotrophic cycle in CA or ovary of D. punctata. The results stress the necessity for validation of reference genes in q-RT-PCR studies in cockroaches.
Highlights
Quantitative RT-polymerase chain reaction (PCR) (q-RT-PCR) is a powerful tool that allows for the large scale analysis of small changes in gene expression
Physiological measurements of the sample animals Oocyte length was determined for each dissected female cockroach used in the q-RT-PCR assays (Figure 1)
Rates of juvenile hormone (JH) release throughout the gonadotrophic cycle steadily rise until four days post final molt, a sharp decline can be seen on day 5 (Figure 1)
Summary
Quantitative RT-PCR (q-RT-PCR) is a powerful tool that allows for the large scale analysis of small changes in gene expression. Since literature suggests that the expression of commonly used reference genes can change in different experimental conditions or tissues and no universally valid reference gene exists, the choice of reference genes cannot be made a priori [4,5,6,7,8]. For this reason, several algorithms were developed to identify the most stably expressed references genes in any given experimental setup [4,6,9]. Many studies aimed at finding the most suitable reference genes in a diverse array of cells, tissues, experimental treatments and different species are being reported
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