The isolation of early nuclear endosperm of Oryza sativa to facilitate gene expression analysis and screening imprinted genes

Quan Kuang, Xiaobo Yu, Xiongbo Peng, Meng-xiang Sun

Since the quality and yield of rice production depends on endosperm development, previous studies have focused on the molecular mechanism that regulates this developmental process. Recently, how this process is epigenetically regulated has become an important topic. However, the gene expression analysis and screening imprinted genes during early endosperm development remain challenging since the isolation of early endosperm has not been possible. Here, we report a procedure for the isolation of endosperm at 24 or 48 HAP (hours after pollination) during the free nuclear stage of endosperm development. This technique allows for rapid and convenient collection of pure free nuclear endosperm. Early endosperm RNA can then be extracted from the isolated endosperm cells using dynabeads. Our results showed that the quality of RNA is satisfactory for gene expression analysis and screening the parental-of-origin specific genes in early endosperm. Thus, we offer a reliable method to overcome one of the major obstacles in the investigation of the molecular mechanisms of early endosperm development. Our approach can be used for accurate gene expression analysis and screening of imprinted genes, and facilitates the confirmation of endosperm-specific gene expression at the very early stages of endosperm development. This method could also be used in other species to collect early free nuclear endosperm.