A method for obtaining high integrity RNA from developing aleurone cells and starchy endosperm in rice (Oryza sativa L.) by laser microdissection

Laser microdissection (LMD) is now becoming a powerful tool to elucidate the spatial profiling of gene expression. However, a remaining difficulty is obtaining high integrity RNA from fixed and embedded tissues in order to obtain accurate and reliable expression analyses. This study aimed to develop methods for the preparation of high integrity RNA from aleurone cells and starchy endosperm of developing rice endosperm by LMD. Acetone or 3:1 ethanol:acetic acid (AA), was used for the fixative and 2% carboxymethyl cellulose (CMC) or paraffin, was used for the embedding reagent. AA and CMC were better for identification of cell types and for recovery of intact RNA, although CMC-embedding did not preserve morphology as well as paraffin-embedding. Quantitative RT-PCR revealed that OsSUT1 mRNA encoding a sucrose transporter was localized in aleurone cells and that SDBE mRNA encoding a starch debranching enzyme (pullulanase) was localized in starchy endosperm. Taken together, these results show that our LMD method is suitable for preparing high integrity RNA from aleurone cells and starchy endosperm in rice. mRNA obtained with this method should help to understand the molecular basis for endosperm development.