Validation of reference genes as internal control for studying viral infections in cereals by quantitative real-time RT-PCR

Abstract

Background: Reference genes are commonly used as the endogenous normalisation measure for the relative quantification of target genes. The appropriate application of quantitative real-time PCR (RT-qPCR), however, requires the use of reference genes whose level of expression is not affected by the test, by general physiological conditions or by inter-individual variability. For this purpose, seven reference genes were investigated in tissues of the most important cereals (wheat, barley and oats). Titre of Barley yellow dwarf virus (BYDV) was determined in oats using relative quantification with different reference genes and absolute quantification, and the results were compared.

Results: The expression of seven potential reference genes was evaluated in tissues of 180 healthy, physiologically stressed and virus-infected cereal plants. These genes were tested by RT-qPCR and ranked according to the stability of their expression using three different methods (two-way ANOVA, GeNorm and NormFinder tools). In most cases, the expression of all genes did not depend on abiotic stress conditions or virus infections. All the genes showed significant differences in expression among plant species. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin (TUBB) and 18S ribosomal RNA (18S rRNA) always ranked as the three most stable genes. On the other hand, elongation factor-1 alpha (EF1A), eukaryotic initiation factor 4a (EIF4A), and 28S ribosomal RNA (28S rRNA) for barley and oat samples; and alpha-tubulin (TUBA) for wheat samples were consistently ranked as the less reliable controls.The BYDV titre was determined in two oat varieties by RT-qPCR using three different quantification approaches. There were no significant differences between the absolute and relative quantifications, or between quantification using GAPDH + TUBB + TUBA +18S rRNA and EF1A + EIF4A + 28S rRNA. However, there were discrepancies between the results of individual assays.

Conclusions: The geometric average of GAPDH, 18S rRNA and TUBB is suitable for normalisation of BYDV quantification in barley tissues. For wheat and oat samples, a combination of four genes is necessary: GAPDH, 18S rRNA, TUBB and EIF4A for wheat; and GAPDH, 18S rRNA, TUBB and TUBA for oat is recommended.

Figure 1

C_q values distribution of candidate reference genes in different species. The values are given as real-time PCR quantification cycle (C_q) values. The boxes represent the lower and upper quartiles with medians; the whiskers illustrate the 10-90 percentiles of the samples. All C_q values significantly differed between species (one-way ANOVA). (W) = wheat samples (60 samples), (B) = barley samples (60 samples), (O) = oat samples (60 samples).

Figure 2

Stability indices (A), and pairwise variation to determine the optimal number of normalisation genes (B) calculated with GeNorm, and stability indices calculated with NormFinder (C). (A) and (C): The stability indices are shown for all species combined, and also barley, wheat and oats individually. The stability of gene expression is inversely proportional to the stability index, so least stable genes have the lowest achieved values and vice versa. (B): The optimal number of genes was determined separately for barley, wheat and oats as well as for all samples combined (all). The recommended cut-off value under which there is no need for inclusion of another gene is 0.15.

Figure 3

BYDV symptoms manifest in two oat varieties compared to a healthy control plant. On the left (A), is a healthy control; in the middle (B) the tolerant cv. Atego, and on the right (C), susceptible cv. Saul.

Figure 4

Titre of BYDV in two oat varieties as determined by three quantification approaches. (A): The relative BYDV titre levels in all oat samples determined by relative quantification using GAPDH, TUBB, TUBA and 18S rRNA (GAPDH group); 28S rRNA, EIF4A and EF1A (EIF4A group); and by absolute quantification with cloned standards (absolute). The absolute numbers were transformed into relative ratios for comparison. The boxes represent the lower and upper quartiles with medians; the whiskers illustrate the 10-90 percentiles of the samples. (B): The relative BYDV titre levels in the two oat cvs. Atego (tolerant) and Saul (susceptible) determined by relative quantification using GAPDH, TUBB, TUBA and 18S rRNA (GAPDH group); 28S rRNA, EIF4A and EF1A (EIF4A group); and by absolute quantification with cloned standards (absolute). The absolute numbers were transformed into relative ratios for comparison. No significant differences were recorded.