Isolation of Arabidopsis nuclei and measurement of gene transcription rates using nuclear run-on assays

被引:51
作者
Folta, Kevin M.
Kaufman, Lon S.
机构
[1] Univ Florida, Dept Hort Sci, Gainesville, FL 32611 USA
[2] Univ Florida, Plant Mol & Cellular Biol, Gainesville, FL 32611 USA
[3] Univ Illinois, Dept Biol, Mol Biol Lab, Chicago, IL 60612 USA
关键词
D O I
10.1038/nprot.2006.471
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:3094 / 3100
页数:7
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