Abstract
Abstract Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a costly disease in rice that threatens global rice production. Gene expression analysis by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) allows the study of the underlying mechanisms of both BB pathogenesis and resistance. In relative quantification, reference genes are often used to normalize the results to remove technical variations allowing the determination of true biological changes in a pilot experiment. However, variations in the expression of these reference genes can lead to erroneous and unreliable results. Thus, choosing the most stable reference genes for any specific experimental condition is of utmost importance in qRT-PCR experiments. Here, we used geNorm, NormFinder, Bestkeeper, Delta-Ct and RefFinder programs and/or methods to analyze the stability of the expression of eleven candidate reference genes namely: 18S ribosomal RNA (18S rRNA), Actin-1 (ACT1), ADP-Ribosylation Factor (ARF), Endothelial differentiation factor (Edf), eukaryotic Elongation Factor-1α (eEF-1α), eukaryotic Initiation Factor-4a (eIF-4a), Profilin 2 (Prof2), Nucleic Acid Binding Protein (NABP), Triosephosphate Isomerase (TI), Ubiquitin 5 (UBQ5) and Ubiquitin 10 (UBQ10) in cDNA samples from BB-susceptible and Xa21-mediated resistant rice cultivars collected at various times after Xoo inoculation. Under our experimental conditions, Edf and TI were the most stable reference genes while the common housekeeping genes 18S rRNA, and UBQ5 were among the least stable genes. Though using either Edf or TI as internal control is adequate for gene expression analysis, we suggest using both genes to normalize the data of qRT-PCR assays for rice subjected to Xoo inoculation.
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call