Abstract
In this report, we present a fluorescence-based approach to the assessment of cellular gene expression and transcription rates. Nuclear run-on was performed by supplying biotin-16-UTP to nuclei, and labeled transcripts were bound to streptavidin-coated magnetic beads. Total cDNA was then synthesized by means of random hexamer primed reverse transcription of captured molecules. To monitor transcript abundance in cDNA, both from nuclear run-on and total RNA, we propose a semiquantitative PCR approach based on the use of fluorescent primers.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Biotinylation*
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Cell Nucleus / genetics*
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Cycloheximide / pharmacology
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DNA Primers
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Drosophila Proteins*
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Fluorescence
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Humans
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Magnetics*
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Microspheres
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Nucleic Acid Hybridization
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Proto-Oncogene Proteins
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Proto-Oncogene Proteins c-ret
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RNA, Messenger / analysis*
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RNA, Messenger / biosynthesis*
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RNA, Messenger / genetics
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RNA, Messenger / isolation & purification
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Receptor Protein-Tyrosine Kinases
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Reverse Transcriptase Polymerase Chain Reaction*
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Transcription, Genetic / drug effects
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Tretinoin / pharmacology
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Tumor Cells, Cultured
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Uridine Triphosphate / analogs & derivatives
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Uridine Triphosphate / metabolism
Substances
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DNA Primers
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Drosophila Proteins
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Proto-Oncogene Proteins
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RNA, Messenger
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Tretinoin
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Cycloheximide
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Proto-Oncogene Proteins c-ret
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Receptor Protein-Tyrosine Kinases
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Ret protein, Drosophila
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Uridine Triphosphate