In Vitro Nuclear Run-on Experiment
The in vitro nuclear run-on experiment is a great way for isolated specific RNA transcript. Also, as shown in the last step, the in vitro nuclear run-on experiment can measure transcription of a gene relative to overall cellular transcription. Together with ion exchange chromatography, which showed that almost all RNA transcripts are translated into protein, this experiment showed that transcriptional control regulates protein production.
| Step | Overview |
|---|---|
| Transcription | After isolation of nuclei, RNAP transcribing beforehand will remain bound to the DNA template and continue. |
| Labeling | Addition of radioactively UTP will label the RNA copies of active genes. |
| ds→ssDNA | Denature dsDNA copies of your gene of interest, then bind the resulting ssDNA to a filter. |
| Hybridization | Incubate the filter with the labeled RNA, thus allowing complementary RNA to bind to the ssDNA on the filter. |
| Elution | Separating hybridized RNA from unhybridized RNA by washing away any labeled ssRNA. |
| Measure | Your gene of interest's relative transcription is the proportion of radioactivity between hybridized and overall RNA. |