|Bull’s-eye: tissue processing improvements for isolating high quality RNA from laser microdissected pathogen infected cells and tissues|
I. J. GIRARD (1), D. Fernando (1), M. G. Becker (1), M. F. Belmonte (1). (1) University of Manitoba, Winnipeg, MB, Canada
Canola is a Canada’s number one oilseed crop, however damage from fungal attacks by the pathogen <i>Leptosphaeria maculans</i> (blackleg) results in millions of dollars in lost productivity every year. The severity of each host-pathogen interaction is governed by complex regulation of transcriptional circuitry that mediates the activation of plant defenses. These regulators are specified in cells at the site of the infection and over time to neighboring cells as the plant responds to the attack. The first step in revealing these processes with a genomics approach is contingent on the isolation of high quality RNA from cells located at and distal to the site of infection, allowing accurate profiles of gene activities for downstream analyses. Laser microdissection is the only method capable of isolating individual cells and tissues from the site of the infection with such a high degree of precision. Tissues of interest are fixed and embedded in paraffin wax to preserve cell structure and RNA, then sectioned for visualization of target cells and microdissected using a Leica LMD 7000 microscope. Optimization of each step of the process is required to overcome RNA degradation from exposure to harsh chemicals like xylenes used in tissue clearing and high temperatures used for wax infiltration. We present quantitative data based on careful optimization of each step in the tissue processing protocol and discuss implications of RNA quality to next generation sequencing strategies.