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 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.
Keller
M A
MA
Molecular Biology Graduate Group, University of Pennsylvania School of Medicine, Philadelphia 19104.
Cassel
D L
DL
Rappaport
E F
EF
McKenzie
S E
SE
Schwartz
E
E
Surrey
S
S
D017398:Alternative Splicing; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D006410:Hematopoiesis; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D016403:Lymphoma, Large B-Cell, Diffuse; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D012334:RNA, Neoplasm / Q000032:analysis / Q000096:biosynthesis; D017452:Receptors, IgG / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D013050:Spectrometry, Fluorescence / Q000379:methods; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D017452:Receptors, IgG