Isolation and in vitro translation of mRNA from rat peritoneal mast cells and rat basophilic leukemia cells

In the absence of any specific literature on the isolation of RNA from mast cells, our initial attempts established that unusual measures would be needed to prepare acceptable yields of high quality RNA from peritoneal mast cells of normal adult rats. Accordingly, we developed procedures for the isolation and characterization of RNA from rat peritoneal mast cells (PMC) and basophilic leukemia cells (RBL). The significant components of the procedures include: separation and removal of mast cell granules to minimize contamination of RNA with proteins and proteoglycans; use of bentonite in phenol extractions; and repetition of extractions and precipitation. The amounts of total RNA extracted from PMC were about 15% of those from RBL, although the percentage mRNA of total RNA in PMC and RBL was similar (1.8 and 2.0%). Ribosomal RNA banding patterns in agarose gel electrophoresis and in vitro translation experiments indicate that the isolated RNA can be employed for analysis of molecular mechanisms of mast cell function and heterogeneity.