Chapter 19 - Analysis of nuclear RNA

Abstract

This chapter presents a comparison of the methods used to assay nuclear RNA, comparing them to the traditional Northern analysis, nuclease protection assays, nuclear run-on (NRO) assay, and the polymerase chain reaction. Two basic approaches are used to study the mechanism of transcription of specific genes. In one approach, the rate of transcription is measured in intact nuclei by the incorporation of labeled precursor nucleotides into RNA transcripts. But the other approach involves the isolation of steady-state nuclear RNA by Northern analysis or nuclease protection assay. The quantity of RNA produced is therefore an indicator of the efficiency of transcription. The degree of success of the NRO assay is entirely dependent on the speed with which nuclei can be isolated from intact cells and radiolabeled. Newer methods have replaced isotopic labeling with the uridine analog 5-bromouridine (BrUrd) which can be sequestered by antibody capture for RT-PCR or next-generation sequencing applications. The relative rate of transcription of several genomic sequences can be evaluated simultaneously by the NRO assay. The NRO assay facilitates the assessment of the transcription rate of individual genes by the elongation of nascent transcripts in the presence of labeled NTP precursor. Both steady-state nuclear RNA and RNA produced by NRO are essential for understanding the processing of the primary transcript. Northern analysis of nuclear RNA yields quantitative data based on signal intensity of discrete bands, but also provides a qualitative component that cannot be discerned by polymerase chain reaction or nuclease protection.