A competitive PCR-based method to measure human fibroblast growth factor receptor 1-4 (FGFR1-4) gene expression.
The four members of the fibroblast growth factor receptor (FGFR) family are cell-surface membrane-spanning tyrosine kinase receptors involved in a wide spectrum of biologic processes. Much evidence also indicates that mutations in FGFR genes result in several craniosynostotic disorders and chondrodysplasias, and that changes in qualitative and quantitative FGFR expression profiles are implicated in tumor induction or progression. Here, we describe a precise and reliable competitive PCR-based assay to evaluate human FGFR1-4 gene expression. A single multispecific synthetic competitive template was designed to amplify FGFR1-4 homologous stretches and constructed to contain FGFR1/FGFR2/FGFR3/FGFR4/GAPDH tandemly arranged forward and reverse primers that allow competition for cDNA-specific primer annealing. The housekeeping GAPDH transcript was utilized as a reference for comparing the expression profiles of different RNA pools. The assay herein described allows the comparison of relative FGFR expression levels, both within a single RNA pool and among multiple RNA pool samples. The major advantages of such a PCR-based approach are its ability to obtain unbiased FGFR mRNA expression patterns and to detect transcripts present in low copy number. Qualitative and semiquantitative analyses of the FGFR1-4 transcript repertoire in mesenchymal- and epithelial-derived primary cell cultures and cell lines demonstrated the utility of such a method to investigate the FGFR1-4 functional role in FGF signal transduction.
Tartaglia
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M
Laboratorio di Metabolisma e Biochimica Patologica, Istituto Superiore di Sanità, Rome, Italy. tartam02@doc.mssm.edu
Fragale
A
A
Battaglia
P A
PA
D017398:Alternative Splicing; D001667:Binding, Competitive; D002460:Cell Line; D002478:Cells, Cultured; D005091:Exons; D020869:Gene Expression Profiling / Q000379:methods*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000096:biosynthesis; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics*
D012333:RNA, Messenger; D017468:Receptors, Fibroblast Growth Factor; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases