Rice Protoplast Isolation and Transfection for Transient Gene Expression Analysis.

Protoplasts are a versatile and powerful cell-based system to study different plant processes in vivo, due to their ability to maintain cell identity and carry out reactions and metabolic processes similar to intact plants. In rice, despite numerous reports, difficulties are encountered in protoplast isolation and transfection. These include insufficient numbers of protoplasts isolated and inefficient transfection. Such difficulties limit the use of this simple yet useful technology. The need to use protoplasts is particularly important when similar experiments may not work in yeast or Pichia, due to differences in functionally essential protein post-translation modifications. In this chapter, we describe a rice protoplast isolation and transfection method.

Reference genes for accurate gene expression analyses across different tissues, developmental stages and genotypes in rice for drought tolerance.

BACKGROUND: Quantitative reverse transcription PCR (qRT-PCR) has been routinely used to quantify gene expression level. This technique determines the expression of a target gene by comparison to an internal control gene uniformly expressed among the samples analyzed. The reproducibility and reliability of the results depend heavily on the reference genes used. To achieve successful gene expression analyses for drought tolerance studies in rice, reference gene selection should be based on consistency in expression across variables. We aimed to provide reference genes that would be consistent across different tissues, developmental stages and genotypes of rice and hence improve the quality of data in qRT-PCR analysis. FINDINGS: Ten candidate reference genes were screened from four ubiquitously expressed gene families by analyzing public microarray data sets that included profiles of multiple organs, developmental stages, and water availability status in rice. These genes were evaluated through qRT-PCR experiments with a rigorous statistical analysis to determine the best reference genes. A ubiquitin isogene showed the best gene expression stability as a single reference gene, while a 3-gene combination of another ubiquitin and two cyclophilin isogenes was the best reference gene combination. Comparison between the qRT-PCR and in-house microarray data on roots demonstrated reliability of the identified reference genes to monitor the differential expression of drought-related candidate genes. CONCLUSIONS: Specific isogenes from among the regularly used gene families were identified for use in qRT-PCR-based analyses for gene expression in studies on drought tolerance in rice. These were stable across variables of treatment, genotype, tissue and growth stage. A single gene and/or a three gene set analysis is recommended, based on the resources available.