Nuclear Run-On Assay to Study Gene Transcription in Vascular Cells

An accurate assessment of gene transcription is important for understanding the mechanism of gene expression. The nuclear run-on assay measures the relative in situ transcription rate of specific genes in intact nuclei (1 ,2 ). It provides information on the synthesis of a specific gene that occurs as a function of cell state, as opposed to a change in mRNA degradation or transport from the nucleus to the cytoplasm. Within a given experimental condition, the nuclear run-on assay can be used to determine the level of transcription for several different genes. The isolated nuclei contain the full transcription machinery for synthesis of mRNA. Therefore, it is regarded as the gold-standard measurement of overall transcriptional activity of a specific promoter. Other methods of measuring gene transcription, e.g., based on RT-PCR or pulse-labeling of nuclear RNA (3 ,4 ), are difficult to perform and are seldom used.