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Methods. 2016 Jul 1;103:77-85. doi: 10.1016/j.ymeth.2016.04.009. Epub 2016 Apr 8.

Single-gene dual-color reporter cell line to analyze RNA synthesis in vivo.

Author information

1
Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States.
2
Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States. Electronic address: Dan.larson@nih.gov.

Abstract

RNA synthesis occurs through the multi-step process of transcription which consists of initiation, elongation, termination, and cleavage of the nascent RNA. In recent years, post-initiation events have attracted considerable attention as regulatory steps in gene expression. In particular, changes in elongation rate have been proposed to alter RNA fate either through changes in RNA secondary structure or recruitment of trans-acting factors, but systematic approaches for perturbing and measuring elongation rate are currently lacking. Here, we describe a system for precisely measuring elongation dynamics for single nascent transcripts at a single gene locus in human cell lines. The system is based on observing the production of fluorescently labeled RNA stem loops which flank a region of interest. The region of interest can be altered using flp recombinases, thus allowing one to study the effects of cis-acting sequences on transcription rate. The dual-color RNAs which are made during this process are exported and translated, thus enabling visualization of each step in gene expression.

KEYWORDS:

Fluorescence; Quantitative; RNA; Single-gene; Single-molecule; Transcription

PMID:
27068658
DOI:
10.1016/j.ymeth.2016.04.009
[Indexed for MEDLINE]

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