Validation of reference genes as internal control for studying viral infections in cereals by quantitative real-time RT-PCR 英文参考文献.docVIP

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Validation of reference genes as internal control for studying viral infections in cereals by quantitative real-time RT-PCR 英文参考文献.doc

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

Jaro?ová and Kundu BMC Plant Biology 2010, 10:146 /1471-2229/10/146 RESEARCH ARTICLE Open Access Validation of reference genes as internal control for Research article studying viral infections in cereals by quantitative real-time RT-PCR Jana Jaro?ová and Jiban K Kundu* 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 quantificat