Nuclear run-on assay using biotin labeling, magnetic bead capture and analysis by fluorescence-based RT-PCR.
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.
Patrone
G
G
Laboratorio di Genetica Molecolare, Istituto G. Gaslini, Università di Genova, Italy. aldamar@tin.it
Puppo
F
F
Cusano
R
R
Scaranari
M
M
Ceccherini
I
I
Puliti
A
A
Ravazzolo
R
R
D019921:Biotinylation; D002467:Cell Nucleus / Q000235:genetics*; D003513:Cycloheximide / Q000494:pharmacology; D017931:DNA Primers; D029721:Drosophila Proteins; D005453:Fluorescence; D006801:Humans; D008280:Magnetics; D008863:Microspheres; D009693:Nucleic Acid Hybridization; D011518:Proto-Oncogene Proteins; D051096:Proto-Oncogene Proteins c-ret; D012333:RNA, Messenger / Q000032:analysis* / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D020794:Receptor Protein-Tyrosine Kinases; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000187:drug effects; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014544:Uridine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism
D017931:DNA Primers; D029721:Drosophila Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D014212:Tretinoin; D003513:Cycloheximide; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C416649:Ret protein, Drosophila; D014544:Uridine Triphosphate