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Abstract
Quantitative real-time polymerase chain reaction (QRT-PCR) has become one of the most
widely used methods for gene expression analysis. However, the expression profile
of a target gene may be misinterpreted due to unstable expression of the reference
genes under different experimental conditions. Thus, a systematic evaluation of these
reference genes is necessary before experiments are performed. In this study, 10 putative
reference genes were chosen for identifying expression stability using geNorm, NormFinder,
and BestKeeper statistical algorithms in 12 different cucumber sample pools, including
those from different plant tissues and from plants treated with hormones and abiotic
stresses. EF1alpha and UBI-ep exhibited the most stable expression across all of the
tested cucumber samples. In different tissues, in addition to expression of EF1alpha
and UBI-ep, the expression of TUA was also stable and was considered as an appropriate
reference gene. Evaluation of samples treated with different hormones revealed that
TUA and UBI-ep were the most stably expressed genes. However, for abiotic stress treatments,
only EF1alpha showed a relatively stable expression level. In conclusion, TUA, UBI-ep,
and EF1alpha will be particularly helpful for reliable QRT-PCR data normalization
in these types of samples. This study also provides guidelines for selecting different
reference genes under different conditions.
Copyright 2009 Elsevier Inc. All rights reserved.