Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions

Abstract

Real-time reverse transcriptase quantitative polymerase chain reaction is the most accurate measure of gene expression in biological systems. The data is analyzed through a process called normalization. Internal standards are essential for determining the relative gene expression in different samples. For this purpose, reference or housekeeping genes are selected based on their constitutive expression across samples. At present, there has not yet been any reference gene identified in any organism that is universally optimal across different tissue types or disease situations. Our goal was to test the regulation of eight housekeeping genes (protein phosphatase 2A, helicase, glyceraldehyse-3-phosphate dehydrogenase, α-tubulin, β-tubulin, actin, elongation factor 1α and 18S ribosomal RNA) in pea plants using the geNorm algorithm. Thirteen samples, including different tissues, treatments and genotypes, were included in this analysis. To validate the determined measure of gene-stability, the gene-specific variation was calculated using different normalization factors. The most non-specific variation was removed when the most stable genes were used for normalization, highlighting the importance of the choice of internal controls in gene expression experiments. The set of reference genes presented here will enable better normalization of transcript levels in pea studies.