Reverse transcriptase in situ polymerase chain reaction for gene expression in rat mast cells and macrophages

Direct reverse transcriptase in situ polymerase chain reaction (RT-in situ PCR) of selected mRNA expression in rat mast cells (MC) and alveolar macrop hages (AM) was optimized. Rat peritoneal mast cells (PMC), rat cultured mas t cells (RCMC), rat bronchoalveolar lavage cells (BALC) or rat cultured alv eolar macrophages (NR8383) were studied for the detection of mRNA for beta- actin, TNF-alpha and/or CD8 alpha. Each type of cell has unique optimal con ditions for RT-in situ PCR. The following parameters were carefully evaluat ed for optimization: protease digestion, DNAse digestion, heparinase digest ion, RT, PCR cycle number and signal development with chromagen. Heparinase digestion was required for PMC mRNA detection because they contain large a mounts of heparin proteoglycan, which is a potent inhibitor of RT and Tag p olymerase enzymes. Only a few PCR cycles were needed to produce a cytoplasm ic signal for mRNA transcripts in RCMC, whereas other types of cells (PMC, BALC and NR8383) needed at least 20 cycles for mRNA detection. The mRNA sig nal in PMC was localized to the perinuclear region, whereas mRNA in other c ell types (RCMC, BALC and NR8383) were detected throughout the cytoplasm. F urthermore, modified Southern blot analysis for TNF-alpha in RCMC treated w ith RT-in situ PCR demonstrated the specificity of amplification product. T he modified and optimized protocols for this procedure were successfully ap plied to detect and localize several mRNA transcripts in rat MC and AM. The approach is valuable and can be used to further study selected gene expres sion in these and other cell types. (C) 1999 Elsevier Science B.V. All righ ts reserved.