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Background  Quantitative 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.    Results  Candidate 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.    Conclusion   Arm  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.

Sequencing and validation of housekeeping genes for quantitative real-time PCR during the gonadotrophic cycle of Diploptera punctata