![]() It is also an important model monocot plant used for genetic and molecular studies. Rice ( Oryza sativa L.) is one of the most important cereal crops, serving as a staple food for almost half of the world's population. Thus, selecting a suitable reference gene and using it under the appropriate conditions is critical to experimental design. For example, the commonly used reference genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin, and tubulin, are not suitable as reference genes under several conditions ( Thellin et al., 1999 Selvey et al., 2001 Lee et al., 2002 Gutierrez et al., 2008). However, most reference genes display constant expression levels only under certain circumstances. Ideally, reference genes should have a constant level of expression across all tissue samples under various experimental conditions ( Suzuki et al., 2000). The use of inappropriate reference genes, on the other hand, may lead to relatively large errors in a significant proportion of samples ( Vandesompele et al., 2002 Huggett et al., 2005). In biological experiments, the selection and use of appropriate reference genes to normalize experimental results is necessary to ensure accuracy and reliability. Therefore, housekeeping genes are usually used as reference genes when comparing the relative expression levels of different samples. These genes are responsible for most basic metabolic processes and they tend to be constitutively expressed. Housekeeping genes refer to the essential genes widely expressed in vivo. In conclusion, the reference proteins selected in this study, and the corresponding antibodies, can be used in qualitative and quantitative analysis of rice proteins.Īntibody-based proteomics, rice ( Oryza sativa L.), reference gene, reference protein, western blotting Introduction Under the present experimental conditions, the lower limits of detection for HSP and eEF-1α proteins in rice were 0.24 ng and 0.06 ng, respectively. Based on the standard curves derived from the antigen–antibody reaction, the concentrations of HSP and eEF-1α proteins in rice leaves were ∼0.12%. Comparison among the profiling of translation and transcription revealed that a correlation existed. ![]() The results indicated that heat shock protein (HSP) and elongation factor 1-α (eEF-1α) were the most constantly expressed among all rice proteins tested throughout all developmental stages, while the proteins encoded by conventional internal reference genes fluctuated in amount. The expression stability of the proteins was analysed using geNorm and Microcal Origin 6.0 software. Western blotting was carried out to detect the expression of target proteins in a set of 10 rice samples representing different rice tissues/organs at different developmental stages. In this study, nine candidate proteins were selected and their specific antibodies were obtained through immunization of rabbits with either recombinant proteins expressed in Escherichia coli or synthesized peptides. To date, no published study has identified and systematically validated reference proteins suitable for the investigation of rice protein expression. In order to obtain reliable expression results in western blotting, information on appropriate reference proteins is necessary for data normalization. Studies of rice protein expression have increased considerably with the development of rice functional genomics. ![]()
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