Figure 1.
Amplification of a specific PCR product with genomic DNA (A) and cDNA (B) as templates on agarose gel (2.0%) using gene-specific primers for each candidate reference gene.
Three replicates of the PCR amplicons with each primer set were loaded; M indicates a100 bp DNA size marker. All primer pairs except CYP2 and LEC amplified a larger size PCR product with DNA template as compared to cDNA template, indicating the position of primer pairs spanning at least one intron.
Figure 2.
The transcriptional profiles of eight individual candidate reference genes (ADH3, ACT11, ATPsyn, CYP2, ELF1B, G6PD, LEC and UBC1) in absolute Cq values over all 31 RNA samples tested.
Figure 3.
Average expression stability and ranking of eight candidate reference genes using geNorm.
All 31 tissue samples set (A), vegetative stage (B), reproductive stage (C), developmental stages (D), viral diseases sample set (E), foliar diseases sample set (F), abiotic stress sample set (G), and different peanut cultivars sample set (H). A lower value of average expression stability (M) indicates more stable expression.
Figure 4.
Determination of the optimal number of reference genes for normalization by pair-wise variation using geNorm.
All 31 tissue samples set (A), vegetative stage (B), reproductive stage (C), developmental stages (D), viral diseases sample set (E), foliar diseases sample set (F), abiotic stress sample set (G) and different peanut cultivars sample set (H). The pairwise variation (Vn/Vn+1) was analyzed between normalization factors NFn and NFn+1 by geNORM program to determine (V<0.15) the optimal number of reference genes.
Figure 5.
Relative quantification of PBNVnp and AtDREB1A genes to validate candidate reference genes of peanut under biotic and abiotic stress conditions.
(A) Expression of PBNVnp gene in infected transgenic peanut leaf sample relatively quantified with candidate reference genes. (B) AtDREB1A gene expression in leaf sample of transgenic (rd29a:AtDREB1A) peanut relatively quantified with candidate reference genes. The relative quantity values were presented after scaling to control samples in both the (PBNVnp and AtDREB1A) cases.