Nuclear run-on
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A nuclear run-on assay is conducted to identify the genes that are being transcribed at a certain point in time. Cell nuclei are isolated rapidly, and incubated with labelled nucleotides and the results are hybridized to a slot blot, which is then exposed to film. It was originally developed by Gariglio et al. (1981) and Brown et al. (1984) (see discussion).
Adding actinomycin-D to the reaction buffer is sufficient to stop transcription, while other toxins, such as α-amanitin, show different effects, making it a good way to assay the effects of these compounds.
This method allows changes in transcription rates to be seen, which often differ from steady-state mRNA levels used in microarrays. This method is obsolete and is not popular due to its "trickiness", the radiolabel and the number of cells needed. Several attempts recently have been made to make this method microarray compatible, but other methods are much more favourable. To have a publication quality blot about 107 nuclei are needed, but 106 nuclei are sufficient to give results: these quantities of cells are higher than for most protocols.
It is often cited that this method is has been surpassed by microarrays, even though it should be noted that this method compares transcription rates and not total transcript expression levels, which differ.
It is incompatible with microarray experiments due to the fact eukaryotic polymerases are more intolerant towards fluorescently labelled or aminoallyl nucleotides, so UTP with phosphorus-32 on the alpha phosphate group is normally used. Cheadle et al (2005) used spotted nylon blots to obtain a high-throughput analysis of T-cell activation.
Alternative microarray methods have recently been developed, mainly PolII RIP-chip: RNA immunoprecipitation of RNA polymerase II with phosphorylated C-terminal domain directed antibodies and hybridization on a microarray slide or chip (the word chip in the name stems from "ChIP-chip" where a special affymetrix genechip was required). No research has been publishing comparing the results obtained with the methods, but each method has its disadvantages and advantages. The main issue on the validity of run on assay results is the presence of artifacts due to the isolation step: some transcripts may stop prematurely others might start out of place.
Another point to add is about the nuclei: There are various methods that require nuclear isolation. The problem is that the nuclear membrane is contiguous with the ER which is rich in protein and cytoplasmic mRNA; some methods require pure nuclear lysate while others, like a nuclear run-on, require "dirty" nuclei with an intact nuclear membrane even if the ER is attached. Some types of cell are notoriously difficult to isolate this way such as lymphocytes, which require a sucrose gradient purification.
See also
References
- Protocol [1]
- http://www.mblab.gla.ac.uk/~julian/dict2.cgi?4568
- Control of gene expression during T cell activation: alternate regulation of mRNA transcription and mRNA stability. Cheadle, C et al. 2005, BMC Genomics 6:75, pp. 75-91.