Isolation of Arabidopsis nuclei and measurement of gene transcription rates using nuclear run-on assays.
Isolation of transcriptionally active nuclei from plant tissues is a fundamental first step in many plant molecular biology protocols. Enriched nuclear fractions may be used in "run-on" assays to measure the rate of transcription for any given gene, adding additional resolution to assays of steady-state transcript accumulation such as RNA-gel blots, RT-PCR or microarrays. The protocols presented here streamline, adapt and optimize existing methods for use in Arabidopsis thaliana. Plant materials are ground in hexylene glycol-based buffers and highly enriched nuclear fractions are obtained using Percoll density gradients. Standard and small-scale protocols are presented, along with a tested method for nuclear run-on assays. The entire process may be completed within 3 days. This capability complements the immense body of steady-state transcript measurements and indirectly identifies instances where message turnover may have a critical and/or primary role in regulating gene expression levels.
Folta
Kevin M
KM
Horticultural Sciences Department and the Plant Molecular and Cellular Biology Program, University of Florida, 1301 Fifield Hall, Gainesville, Florida 32611, USA. kfolta@ifas.ufl.edu
Kaufman
Lon S
LS
D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods*; D002467:Cell Nucleus / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D007553:Isotope Labeling / Q000379:methods*; D009693:Nucleic Acid Hybridization; D014158:Transcription, Genetic