Evaluation and validation of reference genes for normalization of quantitative real-time PCR based gene expression studies in peanut

Abstract

The quantitative real-time PCR (qPCR) based techniques have become essential for gene expression studies and high-throughput molecular characterization of transgenic events. Normalizing to reference gene in relative quantification make results from qPCR more reliable when compared to absolute quantification, but requires robust reference genes. Since, ideal reference gene should be species specific, no single internal control gene is universal for use as a reference gene across various plant developmental stages and diverse growth conditions. Here, we present validation studies of multiple stably expressed reference genes in cultivated peanut with minimal variations in temporal and spatial expression when subjected to various biotic and abiotic stresses. Stability in the expression of eight candidate reference genes including ADH3, ACT11, ATPsyn, CYP2, ELF1B, G6PD, LEC and UBC1 was compared in diverse peanut plant samples. The samples were categorized into distinct experimental sets to check the suitability of candidate genes for accurate and reliable normalization of gene expression using qPCR. Stability in expression of the references genes in eight sets of samples was determined by geNorm and NormFinder methods. While three candidate reference genes including ADH3, G6PD and ELF1B were identified to be stably expressed across experiments, LEC was observed to be the least stable, and hence must be avoided for gene expression studies in peanut. Inclusion of the former two genes gave sufficiently reliable results; nonetheless, the addition of the third reference gene ELF1B may be potentially better in a diverse set of tissue samples of peanut.

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.