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Title: Identification and validation of reference genes for quantitative RT-PCR normalization in wheat
Authors: Paolacci, Anna Rita
Tanzarella, Oronzo A.
Porceddu, Enrico
Ciaffi, Mario
Keywords: Quantitative RT-PCR;Reference genes;Wheat;RT-PCR quantitativa;Geni di riferimento;Frumento
Issue Date: 2009
Publisher: BioMed Central
Source: Paolacci A.R. et al. 2009. Identification and validation of reference genes for quantitative RT-PCR normalization in wheat "BMC Molecular Biology" 10 (11): 1-27
Background: Usually the reference genes used in gene expression analysis have been chosen for
their known or suspected housekeeping roles, however the variation observed in most of them
hinders their effective use. The assessed lack of validated reference genes emphasizes the
importance of a systematic study for their identification. For selecting candidate reference genes
we have developed a simple in silico method based on the data publicly available in the wheat
databases Unigene and TIGR.
Results: The expression stability of 32 genes was assessed by qRT-PCR using a set of cDNAs from
24 different plant samples, which included different tissues, developmental stages and temperature
stresses. The selected sequences included 12 well-known HKGs representing different functional
classes and 20 genes novel with reference to the normalization issue. The expression stability of
the 32 candidate genes was tested by the computer programs geNorm and NormFinder using five
different data-sets. Some discrepancies were detected in the ranking of the candidate reference
genes, but there was substantial agreement between the groups of genes with the most and least
stable expression. Three new identified reference genes appear more effective than the well-known
and frequently used HKGs to normalize gene expression in wheat. Finally, the expression study of
a gene encoding a PDI-like protein showed that its correct evaluation relies on the adoption of
suitable normalization genes and can be negatively affected by the use of traditional HKGs with
unstable expression, such as actin and α-tubulin.
Conclusion: The present research represents the first wide screening aimed to the identification
of reference genes and of the corresponding primer pairs specifically designed for gene expression
studies in wheat, in particular for qRT-PCR analyses. Several of the new identified reference genes
outperformed the traditional HKGs in terms of expression stability under all the tested conditions.
The new reference genes will enable more accurate normalization and quantification of gene
expression in wheat and will be helpful for designing primer pairs targeting orthologous genes in
other plant species.
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ISSN: 1471-2199
DOI: 10.1186/1471-2199-10-11
Appears in Collections:DABAC - Archivio della produzione scientifica

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