Clinical & Experimental Allergy

Degranulation influences heparin-associated inhibition of RT-PCR in human lung mast cells

Authors


Edward S. Schulman, Pulmonary and Critical Care Medicine, Hahnemann University Hospital, M.S.107, Broad & Vine Streets, Philadelphia, PA 19102–1192, USA. E-mail: schulmane@auhs. edu

Abstract

Background The study of gene expression from human lung mast cells (HLMC) has been limited by the ability to reliably detect mRNA transcripts from scant quantities of mast cell RNA contaminated with heparin.

Objective As heparin is granule-associated within the mast cell, we examined the role of degranulation in altering the intrinsic ability of this proteoglycan to inhibit reverse transcription polymerase chain reaction (RT-PCR) from HLMC RNA. We also explored alternative means of RNA isolation to eliminate primary heparin contamination.

Methods Purified HLMC (> 90% pure) were challenged for 2 h with buffer, anti-IgE (3 µg/mL) and/or ionophore A23187 (100 ng/mL) or phorbol 12-myristate 13-acetate (50 ng/mL). Histamine release was measured using a spectroflourometric assay. Following challenge, RNA was isolated by either phenol-chloroform extraction or by nitrocellulose spin column. Parallel samples were either treated with heparinase or placed on ice for 2 h prior to reverse transcription. PCR was performed using primers specific for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Results In each of five studies, GAPDH bands were detected at greater intensity in total cellular RNA (tcRNA) derived from degranulated than from non-degranulated mast cells. However, when examining heparinase-treated tcRNA from the same mast cell samples, the intensity of GAPDH bands normalized between degranulated and non-degranulated cells. When comparing parallel samples of column-purified tcRNA, subsequent treatment of samples with heparinase had no effect on the detection of GAPDH, indicating that endogenous heparin was effectively removed by the technique. Moreover, no variability was noted in GAPDH signal detected from resting vs. degranulated mast cells (n = 3).

Conclusions Degranulation influences the degree of heparin-associated inhibition of RT-PCR in HLMC, and that spin column purification of tcRNA is a time-saving, effective alternative to heparinase pre-treatment.

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