Nuclear run-on assay using biotin labeling, magnetic bead capture and analysis by fluorescence-based RT-PCR

Biotechniques. 2000 Nov;29(5):1012-4, 1016-7. doi: 10.2144/00295st02.

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

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biotinylation*
  • Cell Nucleus / genetics*
  • Cycloheximide / pharmacology
  • DNA Primers
  • Drosophila Proteins*
  • Fluorescence
  • Humans
  • Magnetics*
  • Microspheres
  • Nucleic Acid Hybridization
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-ret
  • RNA, Messenger / analysis*
  • RNA, Messenger / biosynthesis*
  • RNA, Messenger / genetics
  • RNA, Messenger / isolation & purification
  • Receptor Protein-Tyrosine Kinases
  • Reverse Transcriptase Polymerase Chain Reaction*
  • Transcription, Genetic / drug effects
  • Tretinoin / pharmacology
  • Tumor Cells, Cultured
  • Uridine Triphosphate / analogs & derivatives
  • Uridine Triphosphate / metabolism

Substances

  • DNA Primers
  • Drosophila Proteins
  • Proto-Oncogene Proteins
  • RNA, Messenger
  • Tretinoin
  • Cycloheximide
  • Proto-Oncogene Proteins c-ret
  • Receptor Protein-Tyrosine Kinases
  • Ret protein, Drosophila
  • Uridine Triphosphate