Extraction of RNA from dictyostelium.

Abstract

INTRODUCTIONDictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrient conditions are limiting. General principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from cellular and molecular studies of Dictyostelium and have been found to be conserved across all eukaryotes. The availability of a complete genome database and stocks of wild-type and mutant strains make D. discoideum an accessible and powerful model organism. Dictyostelium is amenable to genetic manipulations that require the introduction of DNA into cells, such as gene knockout, overexpression, antisense RNA expression, RNA interference (RNAi)-mediated gene knockdown, and restriction-enzyme-mediated mutagenesis. The extraction of RNA from Dictyostelium is relatively easy because RNA levels are very high in comparison to DNA levels (i.e., ~40 times higher). Certain commercially available kits, such as Trizol (Invitrogen) and RNeasy (QIAGEN) have been used successfully, although lysis conditions need to be adjusted. RNA samples are stable for several years at -80°C in diethylpyrocarbonate (DEPC)-treated H(2)O. For longer-term storage, the RNA pellet can be stored in 100% ethanol at -80°C. Such samples are suitable for Northern blots, reverse transcriptase-polymerase chain reaction (RT-PCR), and microarray analysis of gene expression.