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Reverse transcriptase in situ polymerase chain reaction for gene expression in rat mast cells and macrophages
O Nohara1, M Gilchrist, R E Déry
1Department of Medicine, University of Alberta, Edmonton, Canada.
Abstract:
Direct reverse transcriptase in situ polymerase chain reaction (RT-in situ PCR) of selected mRNA expression in rat mast cells (MC) and alveolar macrophages (AM) was optimized. Rat peritoneal mast cells (PMC), rat cultured mast cells (RCMC), rat bronchoalveolar lavage cells (BALC) or rat cultured alveolar macrophages (NR8383) were studied for the detection of mRNA for beta-actin, TNF-alpha and/or CD8alpha. 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, whereas other types of cells (PMC, BALC and NR8383) needed at least 20 cycles for mRNA detection. The mRNA signal 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-alpha 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.
Insights
Optimized reverse transcriptase in situ polymerase chain reaction (RT-in situ PCR) successfully detects mRNA in rat mast cells and alveolar macrophages. Specific protocols were developed for different cell types, enabling gene expression analysis.
Area of Science:
- Molecular Biology
- Cell Biology
- Immunology
Background:
- Mast cells (MC) and alveolar macrophages (AM) are crucial immune cells.
- Analyzing their gene expression requires precise molecular techniques.
- Previous methods for in situ mRNA detection had limitations.
Purpose of the Study:
- To optimize direct reverse transcriptase in situ polymerase chain reaction (RT-in situ PCR) for detecting specific mRNA.
- To establish unique optimal conditions for RT-in situ PCR in rat mast cells and alveolar macrophages.
- To enable the study of selected gene expression in these immune cells.
Main Methods:
- Direct RT-in situ PCR was optimized for rat peritoneal mast cells (PMC), cultured mast cells (RCMC), bronchoalveolar lavage cells (BALC), and cultured alveolar macrophages (NR8383).
- Key parameters optimized included protease, DNAse, and heparinase digestion, RT conditions, PCR cycle number, and signal development.
- Modified Southern blot analysis was used to confirm the specificity of amplification products for TNF-alpha.
Main Results:
- Optimal RT-in situ PCR conditions varied significantly between cell types.
- Heparinase digestion was essential for PMC due to heparin proteoglycan inhibition.
- RCMC required fewer PCR cycles (few) compared to PMC, BALC, and NR8383 (≥20) for mRNA detection.
- mRNA localization differed: perinuclear in PMC, cytoplasmic in RCMC, BALC, and NR8383.
- Specificity of amplification was confirmed using Southern blot analysis.
Conclusions:
- Optimized RT-in situ PCR protocols effectively detect and localize mRNA in rat MC and AM.
- The method's adaptability allows for the study of diverse gene expression patterns in various cell types.
- This technique provides a valuable tool for advancing research in cellular gene expression analysis.
