Ž . Journal of Immunological Methods 226 1999 147–158 Reverse transcriptase in situ polymerase chain reaction for gene expression in rat mast cells and macrophages O. Nohara, M. Gilchrist, R.E. Dery, G.R. Stenton, N.S. Hirji, A.D. Befus ) ´ Glaxo-Heritage Asthma Research Laboratory, Room 574 HMRC, Department of Medicine, UniÕersity of Alberta, Edmonton, Alberta, T6G 2S2, Canada Accepted 2 April 1999 Abstract Ž . Direct reverse transcriptase in situ polymerase chain reaction RT-in situ PCR of selected mRNA expression in rat mast Ž . Ž . Ž . cells MC and alveolar macrophages AM was optimized. Rat peritoneal mast cells PMC , rat cultured mast cells Ž . Ž . Ž . RCMC , rat bronchoalveolar lavage cells BALC or rat cultured alveolar macrophages NR8383 were studied for the detection of mRNA for b-actin, TNF-a andror CD8 a . Each type of cell has unique optimal conditions for RT-in situ PCR. The following parameters were carefully evaluated for optimization: protease digestion, DNAse digestion, heparinase digestion, RT, PCR cycle number and signal development with chromagen. Heparinase digestion was required for PMC mRNA detection because they contain large amounts of heparin proteoglycan, which is a potent inhibitor of RT and Taq polymerase enzymes. Only a few PCR cycles were needed to produce a cytoplasmic signal for mRNA transcripts in RCMC, Ž . whereas other types of cells PMC, BALC and NR8383 needed at least 20 cycles for mRNA detection. The mRNA signal Ž . in PMC was localized to the perinuclear region, whereas mRNA in other cell types RCMC, BALC and NR8383 were detected throughout the cytoplasm. Furthermore, modified Southern blot analysis for TNF-a in RCMC treated with RT-in situ PCR demonstrated the specificity of amplification product. The modified and optimized protocols for this procedure were successfully applied 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 expression in these and other cell types. q 1999 Elsevier Science B.V. All rights reserved. Keywords: RT-in situ PCR; Mast cell; Macrophage; Heparinase; TNF-a; CD8 AbbreÕiations: RT-PCR, Reverse transcriptase polymerase chain reaction; ISH, In situ hybridization; MC, Mast cells; PMC, Peritoneal mast cells; RCMC, Rat cultured mast cells; TNF-a , Tumor necrosis factor a ; BALC, Bronchoalveolar lavage cells; AM, Alveolar macrophages; SD, Sprague–Dawley; BN, Brown– Norway; HBTS, Hepes-buffered Tyrode’s solution; PBS, Phos- phate-buffered saline; DEPC, Diethylpyrocarbonate; NBT, 4-Nitro blue tetrazolium chloride; BCIP, 5-Bromo-4-chloro-3-indolyl phosphate; SSC, Saline sodium citrate ) Corresponding author. Tel.: q1-780-492-1909; Fax: q1-780- 492-5329; E-mail: dean.befus@ualberta.ca 1. Introduction Direct cellular localization of a DNA or RNA Ž . target can be achieved by in situ hybridization ISH . However, the relatively high detection threshold of ISH limits its usefulness. At least 10 to 20 DNA or RNA copiesrcell are needed for the target to be Ž . detectable by conventional ISH Nuovo, 1997 . Mes- 0022-1759r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0022-1759 99 00061-7