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 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.
Department of Medicine, University of Alberta, Edmonton, Canada.
O
O
Nohara
M
M
Gilchrist
R E
RE
Déry
G R
GR
Stenton
N S
NS
Hirji
A D
AD
Befus
D000818:Animals; D016827:CD8 Antigens / Q000235:genetics*; D002460:Cell Line; D015870:Gene Expression; D016676:Macrophages, Alveolar / Q000378:metabolism*; D008297:Male; D008407:Mast Cells / Q000378:metabolism*; D012333:RNA, Messenger; D051381:Rats; D011914:Rats, Inbred BN; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012996:Solutions; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D016827:CD8 Antigens; D012333:RNA, Messenger; D012996:Solutions; D014409:Tumor Necrosis Factor-alpha