Isolation of Arabidopsis nuclei and measurement of gene transcription rates using nuclear run-on assays
Isolation of Arabidopsis nuclei and measurement of gene transcription rates using nuclear run-on assays
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DOI：
10.1038/nprot.2006.471
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Kaufman, Lon S.
中科院分区：
文献类型：
--
作者：
Folta, Kevin M.;Kaufman, Lon S.
从植物组织中分离转录活性核是许多植物分子生物学方案中的基本第一步。富集的核分数可用于“跑步”测定法以测量任何给定基因的转录速率，并在稳态转录物积累（例如RNA凝胶印迹，RT-PCR或微阵列）的测定中增加了额外的分辨率。此处介绍的协议在此处呈现的协议，适用于拟南芥的现有方法，用于拟南芥材料。使用珀托尔密度梯度获得了分数，并提出了小规模的方案，并在3天内完成了核跑步测定的测试方法。
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.