Optimization of the isolation and effective use of mRNA from rat mast cells.
To define the molecular regulation of mast cell phenotype and function optimized procedures must be available to study mRNA from mast cells freshly isolated from tissues. However, rat peritoneal mast cells (PMC) contain large amounts of the proteoglycan heparin, and unfortunately, this molecule which is a potent inhibitor of reverse transcriptase (RT) and Taq polymerase and thus RT-PCR, copurifies with RNA. Here we describe an optimized protocol for extracting and amplifying RNA from rat PMC. Mast cells were isolated from rat peritoneum and a method modified from that of Chomczynski and Sacchi (1987) was used to extract the RNA. Following the removal of heparin by heparinase digestion, first strand cDNA synthesis was primed with oligo-dT and the resulting cDNA was quantified by rapid paper chromatography. The use of a detection system for the reverse transcription reaction ensured that the production of cDNA had occurred and allowed subsequent PCR testing to be optimal. cDNA thus produced can be used to detect relatively specific (histidine decarboxylase) and non-specific (beta-actin) mast cell products. Our PCR studies have shown a 300-fold increase in sensitivity over RNA processed by other methods.
Gilchrist
M
M
Department of Medicine, University of Alberta, Edmonton, Canada.
MacDonald
A J
AJ
Neverova
I
I
Ritchie
B
B
Befus
A D
AD
D000818:Animals; D002478:Cells, Cultured; D006493:Heparin / Q000032:analysis; D019763:Heparin Lyase; D008297:Male; D008407:Mast Cells / Q000737:chemistry*; D008722:Methods; D010529:Peritoneal Cavity / Q000166:cytology; D016133:Polymerase Chain Reaction / Q000379:methods; D011133:Polysaccharide-Lyases / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification*; D051381:Rats; D017207:Rats, Sprague-Dawley
D012333:RNA, Messenger; D006493:Heparin; D011133:Polysaccharide-Lyases; D019763:Heparin Lyase