Nuclear Run‐on Assays

Abstract

The nuclear run‐on assay is used to measure the transcriptional activity of selected endogenous genes. Nuclei are isolated from appropriate cells using techniques that keep engaged RNA polymerase complexes bound to genomic DNA. Subsequent incubation with the four ribonucleotide triphosphates, one of which is radiolabelled, allows polymerase complexes to progress several hundred base pairs along the genome, producing short, radioactive RNA molecules. After purification, the RNA species of interest are detected by hybridization to appropriate DNA sequences immobilized on a membrane. The amount of specifically hybridized RNA is proportional to the number of engaged polymerase complexes and therefore reflects the transcriptional activity of the gene in the intact cell.

Keywords: gene transcription; gene expression; nuclei; slot blots; RNA labelling

Figure 1.

Autoradiograph of a typical nuclear run‐on experiment. In this experiment two identical filter strips (prepared as in Step 2) have been probed with 32P‐labelled RNA derived from nuclei from a single cell line grown under two different conditions, A and B. The filters had been loaded with single‐stranded cDNA sequences from the sense and antisense strand from three genes of interest (1, 2 and 3) plus the control gene GAPDH. Further controls included wells loaded with the plasmid vector and water.

Figure 2.

Effect of pre‐treating the nuclei with RNase A. The transcription reaction was carried out on two aliquots of nuclei with an without pre‐treatment with RNase A as indicated. Approximately 50 000 cpm of labelled RNA were then separated on a formaldehyde denaturing agarose gel and blotted to membrane for autoradiography. The two parts of the figure are derived from separate gels, hence the position of the 28S and 18S rRNA bands differ and the intensities of the autradiographs cannot be directly compared. The difference in size complexity of the two RNA probes is however clearly evident.

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References

Roberts S and Bentley D (1992) Distinct modes of transcription read through or terminate at the c‐myc attenuator. The EMBO Journal 11: 1085–1093.

Sierra F, Tian JM and Schibler U (1993) In vitro transcription with nuclear extracts from differentiated tissues. In: Hames BD and Higgins SJ (eds) Gene Transcription – A Practical Approach, pp. 125–152. Oxford, UK: IRL Press.

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How to Cite close
Bates, N, and Hurst, H(Oct 2002) Nuclear Run‐on Assays. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0003785]