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.

This method allows changes in transcription rates to be seen, which often differ from steady-state mRNA levels used in microarrays. Unfortunately this method is not very popular due to the radiolabel and the number of cells needed. Eukaryotic polymerases are more intolerant towards fluorescently labelled or aminoallyl nucleotides, so UTP with phosphorus-32 on the alpha phosphate group is normally used. To have a publication quality blot about 10⁷ nuclei are needed, but 10⁶ nuclei are sufficient to give good results. Adding actinomycin-D to the reaction buffer is suffient 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.

[edit] Nuclear Isolation

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.