This Article Full Text (PDF) References Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Keller, M A Articles by Surrey, S Search for Related Content PubMed PubMed Citation Articles by Keller, M A Articles by Surrey, S Social Bookmarking                   What's this?

Fluorescence-based RT PCR analysis: determination of the ratio of soluble to membrane-bound forms of Fc gamma RIIA transcripts in hematopoietic cell lines.

Molecular Biology Graduate Group, University of Pennsylvania School of Medicine, Philadelphia 19104.

Abstract

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 Fc gamma RIIA transcripts, Fc gamma RIIa1 and Fc gamma RIIa2, 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 (Fc gamma RIIa2/Fc gamma RIIa1) in several hematopoietic cell lines. The ratio varies from 0.70 (41% Fc gamma RIIa2) in the erythroleukemic cell line HEL, to 0.14 (12% Fc gamma RIIa2) in the monocytic cell line U937, to 0.07 (6% Fc gamma RIIa2) 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.

CiteULike

Connotea

Del.icio.us

Digg

Reddit

Technorati