Defining reference genes for qPCR normalization to study biotic and abiotic stress responses in Vigna mungo

Expression of ACT, EF1A; H2A, EF1A, ACT and 18S, TUB showed stability under MYMIV, salinity and drought stress, respectively; these are recommended as reference genes for qPCR normalization in Vigna mungo. Accurate gene expression profiling through qPCR depends on selection of appropriate reference gene(s) for normalization. Due to lack of unanimous internal standard, suitable constitutively expressed reference genes are selected that exhibit stable expression under diverse experimental conditions. In this communication, a comparative evaluation of stability among seven V. mungo genes encoding actin (ACT), histone H2A (H2A), elongation factor 1-alpha (EF1A), 18S rRNA (18S), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), cyclophilin (CYP) and tubulin (TUB) under biotic (MYMIV) and abiotic (drought and salinity) stress conditions has been attempted. Specificity and amplification efficiency for each primer pair were verified; however, cumulative assessment of their accumulated transcripts revealed no uniformity. Therefore, individual stability and suitability of these seven candidates have been assessed in silico, by two widely used algorithms, geNorm and Normfinder. Based on the computed results, high stability was obtained for ACT and EF1A during MYMIV stress, while H2A, EFIA and ACT were found to be most suitable in salinity stress experiments and TUB and 18S during drought treatments. Combinations of ACT/TUB or ACT/EFIA were recommended for their use in the pooled analysis, while expression of 18S and CYP showed greater variations and therefore considered unsuitable as reference genes. Additionally, precise quantification of the target gene VmPRX under these stresses was shown to be a function of reference genes' stability, which tends to get affected when normalized with the least stable genes. Hence, use of these normalizers will facilitate accurate and reliable analyses of gene expression in V. mungo.