The purpose of a nuclear run-on transcription experiment is to measure the ongoing transcription activity in the cell nucleus by labeling and quantifying the newly synthesized RNA transcripts. This technique provides information about the transcription rates of specific genes and can help determine the regulation of gene expression.
In a nuclear run-on transcription experiment, isolated cell nuclei are incubated in the presence of labeled nucleotides (e.g., radioactive or fluorescently labeled nucleotides) that can be incorporated into the nascent RNA transcripts. The labeled nucleotides are only incorporated into the newly synthesized RNA molecules, allowing for the specific detection and quantification of active transcription.
By comparing the signal obtained from the labeled RNA transcripts with an appropriate control, such as total cellular RNA or a non-transcriptionally active sample, researchers can determine the relative transcriptional activity of specific genes. This technique can be used to investigate changes in gene expression under different conditions, identify transcriptional regulatory elements, study the effects of mutations or treatments on transcription rates, and explore the dynamics of transcriptional regulation.
On the other hand, run-off transcription is a different technique that measures the completion of transcription of pre-existing RNA molecules. In a run-off transcription experiment, the cells are treated with inhibitors that halt transcription initiation while allowing the elongation of already initiated transcripts to continue. After the treatment, the RNA molecules are isolated and analyzed to determine the length and abundance of the transcripts.
Unlike nuclear run-on transcription, run-off transcription does not measure ongoing transcription rates. Instead, it provides information about the status and processing of pre-existing RNA molecules. Run-off transcription experiments can be useful for studying transcription termination, RNA processing events, or assessing the stability of specific RNA species.
In summary, the nuclear run-on transcription experiment measures ongoing transcription activity by labeling and quantifying newly synthesized RNA transcripts, while run-off transcription experiments focus on the completion and processing of pre-existing RNA molecules.