Laser Microdissection of Plant Tissues

Abstract

Laser microdissection (LMD) is one of the most effective methods used to isolate cells from heterogeneous tissues. Global or targeted profiling of specific cells is easily achievable for the majority of plant and animal systems. However, for these methods to be successful, tissues must be processed to preserve cellular detail for the identification of target cell types using general light microscopy in addition to the recovery of target molecules (RNA, DNA, or proteins). Paraffin as an embedding medium is one of the few means by which tissues can be prepared for LMD. The main advantage of paraffin embedding compared with other methods is that it maintains anatomical integrity of plant cells, allowing for the identification of target cell types that may have subtle morphological differences. Using this technique, tissues are dehydrated, infiltrated, and embedded with paraffin wax, sectioned with a microtome, mounted on slides with a polyethylene naphthalate membrane, and finally de-paraffinized with xylenes. All tissues can be processed using this general procedure; however, the duration of each step must be optimized depending on the cellular features of the tissue of interest. Here, we present optimized tissue processing protocols for paraffin-embedded Brassica napus seed and leaf tissues for LMD for RNA profiling experiments. Paraffin-embedded LMD has been applied to multiple RNA profiling experiments such as microarray analyses, directed quantitative PCR, small RNA sequencing, methylome sequencing, and RNA sequencing. The combination of LMD with the versatility of its downstream applications makes it a powerful technology for the high-resolution study of cellular bioprocesses.