Nuclear Run-On AssayNuclear run-on assays are transcription assays designed to look at the genes being transcribed in a cell nucleus at a specific time. The principle involved is that, when you lyse a cell and retrieve the entact nuclei, the isolated nuclei contain transcription complexes stalled on the DNA template due to acute loss of ribonucleotide substrates. Transcription has thus been halted at the point in time where the nuclei were removed, but can be started up again in vitro with the addition of new ribonucleotides.
By using radiolabled nucleotides, the experimenter can allow transcription to finish, and then observe via autoradiography which mRNA transcripts were produced from the stalled RNA polymerase reactions and thus which genes were being transcribed at the time the cells were lysed. By comparing the amount of gene-specific radiolabled RNA synthesized in one nuclei prep with another you can get an idea of the transcriptional initiation events in the cells of interest. Generally, purified mRNA is hybridized to cDNA on a membrane and analyzed via autoradiography. For example, you could treat one group of cells with a signaling molecule such as a growth factor and then perform nuclear run on for these cells and another group of untreated cells, then compare the radiolabled mRNA produced, looking specifically at genes expected to be upregulated. In this protocol, one dish of confluent cells in culture would receive the growth factor, while another identical dish would not. Both dishes would then be harvested, the cells would be lysed and the nuclei purified. It is important to isolate nuclei free of cytoplasmic material and cell membranes and not to damage the nuclei in the process, as both can lead to poor incorporation of radiolabled nucleotides. The difficulty in this varies largely with cell type, but is generally the biggest potential obstacle in the assay. After isolation, the nuclei are incubated with 32P-UTPs and transcription is allowed to finish. RNA is then purified from the nuclei and hybridized to cDNA on a nitrocellulose membrane. cDNA should be from the genes of interest along with positive and negative control genes (expected high or low expression from both groups of cells), and autoradiography should give a good sense of the level of transcription relative to the untreated control.
This procedure can be difficult to perform with certain cell types due to difficulty isolating nuclei. Lymphocytes, for example, tend to have fragile nuclei that are easily damaged during isolation. Furthermore, it may be possible that new RNA synthesis may be initiated in the isolated nuclei during the run-on reaction, leading to radiolabled mRNA that does not represent "stalled transcription". Use of this assay to assess gene expression levels has largely been supplanted by the use of microarray analysis, hover this assay is still useful in assessing whether changes in mRNA levels are a function of transcription or of subsequent RNA degradation or transport.
Editor(s): John Bracht
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