Protocol

Isolation of RNA and DNA from Biofilm Samples Obtained by Laser Capture Microdissection Microscopy

Department of Microbiology, Montana State University, Bozeman, MT 59717, USA

¹Corresponding author (umbfm{at}montana.edu)

INTRODUCTION

The metabolic activities of bacteria growing in biofilms result in spatial gradients of oxygen, nutrients, and waste products. Because bacteria respond to local environmental conditions through changes in gene expression, mRNA levels of individual genes may vary spatially among bacteria within the biofilm. This article describes an approach to isolate RNA for quantification from cells at localized sites within biofilms. Biofilm thin sections are generated by embedding biofilms in cryoembedding resin, freezing the embedded biofilms on dry ice, and cutting with a cryomicrotome. The sections are placed on membrane-coated microscope slides and maintained on dry ice. Laser capture microdissection microscopy (LCMM) is used to dissect small subsets of cells at different regions within the biofilms, and RNA is extracted from the samples using either hot phenol or TRI reagent. A TRI reagent-based DNA extraction method is also presented.