Standardization of reference genes for real-time PCR analysis of Vigna radiata L. under Agrobacterium tumefaciens infection

Abstract

Gene expression analysis using real-time polymerase chain reaction (PCR) requires the validation of a stable reference gene for normalization. Previously reported candidates may not be suitable for novel crops. Vigna radiata is a protein-rich legume crop used in many countries across the world. As this crop is recalcitrant to transformation such as most legumes, we were interested in V. radiata-Agrobacterium interaction studies. Toward this end, we wanted to identify a suitable reference for normalization under Agrobacterium-infections conditions. We selected seven candidate genes (Actin, Tubulin, Glyceraldehyde 3-phosphate dehydrogenase, Ubiquitin 10, Cyclophilin, 18S Ribosomal RNA, and F-box protein) and performed temporal-expression analysis using real-time PCR at 3 h, 24 h, and 4-day post-infection. When their Ct values were compared using the routinely used four algorithms, Normfinder, geNorm, Bestkeeper, and RefFinder, each suggested a different candidate gene. We narrowed-down to Cyclophilin by taking the statistical approach of using analysis of variance and Tukey honestly significant difference. Thus, our report signifies the use of additional statistical analyses in case of situations of discrepancies due to variable suggestions by software.