DETECTION OF MOUSE MAST CELL-ASSOCIATED PROTEASE MESSENGER-RNA - HEPARINASE TREATMENT GREATLY IMPROVES RT-PCR OF TISSUES CONTAINING MAST-CELL HEPARIN

The reverse transcriptase polymerase chain reaction (RT-PCR) procedure is markedly inhibited in specimens of blood that contain commercial h eparin as an anticoagulant or in cell preparations containing rat or m ouse peritoneal mast cells. However, it was not known whether the leve ls of endogenous, mast cell-associated heparin that are present in som e mammalian tissues are sufficient to interfere with the use of RT-PCR in these settings. We show that RT-PCR detects little or no mRNA tran scripts for either mast cell-associated products, such as mouse mast c ell-associated protease-2 or -4 (MMCP-2 or MMCP-4) or mast cell carbox ypeptidase A, or for mast cell-nonspecific products, such as glycerald ehyde 3-phosphate dehydrogenase, in routinely prepared specimens of ce lls or tissues that include populations of heparin-containing mast cel ls. However, signals for mast cell-associated or mast cell-nonspecific transcripts can be readily detected ill such specimens if they are tr eated with heparinase before RT-PCR. RT-PCR after heparinase treatment appears to represent an extremely sensitive method for detecting mast cell-associated transcripts in tissue specimens, permitting the ident ification of transcripts for mast cell-specific proteases in the skin of genetically mast cell-deficient WBB6GF(1)-W/(V) mice, a tissue that contains few or no mast cells according to histological analysis.