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, 809, 505-17

Array-based Nuclear Run-On Analysis

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Array-based Nuclear Run-On Analysis

Jinshui Fan et al. Methods Mol Biol.

Abstract

There is extensive evidence that posttranscriptional mechanisms of gene regulation, such as mRNA turnover, critically affect the patterns of expressed mRNAs. Conventional microarray analysis measures steady-state messenger RNA (mRNA) levels, which represents the dynamic balance between new transcription and mRNA degradation. Accordingly, only de novo transcription can accurately reflect the temporal and spatial events of transcriptional regulation. In this chapter, we describe a recently reported method to study transcription systematically. It involves the genome-wide labeling of nascent transcripts using nonradioactive modified nucleotides, their isolation for amplification, and their hybridization and analysis using commercial microarrays.

Figures

Figure 1
Figure 1
Schematic of the protocol Fan et al.,
Figure 2
Figure 2
Detection of full-length nascent transcripts by the ANRO method. Affymetrix exon arrays were used to detect the 91 exons of the PRKDC gene spanning 190 KB of genomic DNA. As the PRKDC gene transcription is under control by the Myc promoter, PRKDC transcription rates and mRNA abundance were compared under conditions of high Myc expression (Myc ON) and no Myc expression (Myc OFF). Graphs represent the expression intensity of each exon using nascent pre-mRNA prepared by the ANRO method (A) and using total RNA prepared using conventional methods (B). As shown, all exons are represented in ANRO arrays, indicating that the nascent PRKDC pre-mRNA is labeled evenly throughout the entire transcript (A). Moreover, the label incorporation in the newly transcribed PRKDC pre-mRNA (A) mirrors the abundance of each exon in the mature PRKDC mRNA (B). When comparing Myc ON vs. Myc OFF, PRKDC pre-mRNA levels are induced by 2.71-fold (A), PRKDC mRNA levels by 3.71-fold. FDR, false discovery rate. Fan et al.,

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