FLUORESCENCE-BASED RT PCR ANALYSIS - DETERMINATION OF THE RATIO OF SOLUBLE TO MEMBRANE-BOUND FORMS OF FC-GAMMA-RIIA TRANSCRIPTS IN HEMATOPOIETIC-CELL LINES

We have developed a fluorescence-based RT PCR assay for determination of the ratio of two alternatively spliced transcripts in different cell types. Fluorescence detection, by an automated DNA sequencer, allows enhanced sensitivity and ease of data processing. PCR products are fluorescently tagged using a dye-labeled oligonucleotide primer during the PCR reaction. Assay conditions were first defined so that fluorescence intensity of the PCR products was linear with respect to input RNA and exponential relative to PCR cycle number. Sensitivity and reproducibility of detection were evaluated with serial dilutions of RT PCR reactions. We have applied this assay to an analysis of the lineage-specific expression of two human FcgammaRIIA transcripts, FcgammaRIIa1 and FcgammaRIIa2, in different hematopoietic cell lines. Previously, we noted that when standard RT PCR conditions are used with primers that bracket the TM exon, the pattern of expression of these transcripts as assessed by ethidium bromide staining of agarose gels varied in different hematopoietic cell lineages. Using the fluorescence-based RT PCR method, we now confirm our previous findings and quantitate transcript ratios (FcgammaRIIa2/FcgammaRIIa1) in several hemapoietic cell lines. The ratio varies from 0.70 (41% FcgammaRIIa2) in the erythroleukemic cell line HEL, to 0.14 (12% FcgammaRIIa2) in the monocytic cell line U937, to 0.07 (6% FcgammaRIIa2) in the multipotential cell line K562. This fluorescent RT PCR method provides a general approach to quantitating mRNA levels and ratios of PCR products in other gene systems.