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Background  Soybean pathogens and pests reduce grain production worldwide. Biotic interaction cause extensive changes in plant gene expression profile and the data produced by functional genomics studies need validation, usually done by quantitative PCR. Nevertheless, this technique relies on accurate normalization which, in turn, depends upon the proper selection of stable reference genes for each experimental condition. To date, only a few studies were performed to validate reference genes in soybean subjected to biotic stress. Here, we report reference genes validation in soybean during root-knot nematode ( Meloidogyne incognita ) parasitism and velvetbean caterpillar ( Anticarsia gemmatalis ) attack.    Findings  The expression stability of nine classical reference genes ( GmCYP2 ,  GmELF1A ,  GmELF1B ,  GmACT11 ,  GmTUB ,  GmTUA5 ,  GmG6PD ,  GmUBC2  and  GmUBC4 ) was evaluated using twenty-four experimental samples including different organs, developmental stages, roots infected with  M. incognita  and leaves attacked by  A. gemmatalis . Two different algorithms ( geNorm  and  NormFinder ) were used to determine expression stability.  GmCYP2  and  GmUBC4  are the most stable in different organs. Considering the developmental stages,  GmELF1A  and  GmELF1B  genes are the most stable. For spatial and temporal gene expression studies, normalization may be performed using  GmUBC4 ,  GmUBC2 ,  GmCYP2  and  GmACT11  as reference genes. Our data indicate that both  GmELF1A  and  GmTUA5  are the most stable reference genes for data normalization obtained from soybean roots infected with  M. incognita , and  GmCYP2  and  GmELF1A  are the most stable in soybean leaves infested with  A. gemmatalis .    Conclusions  Future expression studies using nematode infection and caterpilar infestation in soybean plant may utilize the reference gene sets reported here.

Validation of reference genes aiming accurate normalization of qPCR data in soybean upon nematode parasitism and insect attack