Isolation of rice sperm cells for transcriptional profiling

Abstract

The male germline of flowering plants displays unexpectedly divergent transcriptional profiles compared to other cell types and tissues of plants. As these are among the smallest cells, and are harbored within pollen, isolating a pure collection of germline RNA presents unusual challenges. The sperm cells of rice represent a particularly challenging subject for study as the pollen are unusually short lived upon release from the anther, and the marker gene sequences that make FACS possible in Arabidopsis have not yet been introduced into rice. The purity of the germline samples requires careful collection because of the limited amount of material available and potential contamination by other nearby tissues, pollen, and RNases. A discontinuous Percoll density gradient centrifuge was developed to isolate and obtain enough rice sperm cells for RNA-seq or microarray analysis.