Selection of housekeeping genes for normalization by real-time RT–PCR: Analysis of Or-MYB1 gene expression in Orobanche ramosa development

• Published in: Analytical biochemistry Vol. 379; no. 2; pp. 176 - 181
• Main Authors: González-Verdejo, C.I., Die, J.V., Nadal, S., Jiménez-Marín, A., Moreno, M.T., Román, B.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-08-2008
• Subjects: Endogenous control genes; Gene Expression Profiling; Gene Expression Regulation, Plant; Genes, Plant - genetics; MYB transcription factor; Orobanche - genetics; Orobanche - growth & development; Parasitic plant; Real-time RT–PCR; Reference Standards; Reverse Transcriptase Polymerase Chain Reaction - methods; Reverse Transcriptase Polymerase Chain Reaction - standards; RNA, Messenger - genetics; RNA, Messenger - metabolism; Sensitivity and Specificity; Time Factors; Endogenous control genes; Real-time RT–PCR; MYB transcription factor; Parasitic plant
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2008.05.003

Real-time PCR has become the method of choice for accurate and in-depth expression studies of candidate genes. To avoid bias, real-time PCR is referred to one or several internal control genes that should not fluctuate among treatments. A need for reference genes in the parasitic plant
Orobanche ramosa has emerged, and the studies in this area have not yet been evaluated. In this study, the genes
18S rRNA, Or-act1, Or-tub1, and
Or-ubq1 were compared in terms of expression stability using the BestKeeper software program. Among the four common endogenous control genes,
Or-act1 and
Or-ubq1 were the most stable in
O. ramosa samples. In parallel, a study was carried out studying the expression of the transcription factor
Or-MYB1 that seemed to be implicated during preinfection stages. The normalization strategy presented here is a prerequisite to accurate real-time PCR expression profiling that, among other things, opens up the possibility of studying messenger RNA levels of low-copy-number-like transcription factors.