Nuclear run on assay compare 4su

. 2009 Nov;2009(11):pdb.prot5329.

Índice Show

Nuclear run-on assay
Similar articles
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
What is a nuclear run
What are nascent transcripts?
What is nascent RNA sequencing?
Do all mRNA have poly A tail?

doi: 10.1101/pdb.prot5329.

PMID: 20150068
DOI: 10.1101/pdb.prot5329

Nuclear run-on assay

Stephen T Smale. Cold Spring Harb Protoc. 2009 Nov.

Abstract

The nuclear run-on assay was developed as a method for establishing that the transcription initiation rate contributes to the regulated expression of mammalian genes. The difference between monitoring gene expression by the nuclear run-on assay versus most other assays is that the nuclear run-on assay provides a measure of the frequency of transcription initiation and is largely independent of the effects of RNA stability. It can also be used to determine whether polymerase pausing or attenuation contributes to gene regulation. Briefly, the nuclear run-on assay begins with samples of cells that contain different steady-state amounts of the mRNA or protein of interest. The cells are chilled, and the plasmid membranes are permeabilized or lysed. These steps result in polymerase pausing. The nuclei are then incubated for a short time at 37 degrees C in the presence of nucleoside triphosphates (NTPs) and radiolabeled uridine 5'-triphosphate (UTP). New transcripts are not initiated during this incubation, but the radiolabeled nucleotide becomes incorporated into transcripts that were being synthesized when the cells were first chilled and lysed. The number of nascent transcripts on the gene at the time of chilling is thought to be proportional to the frequency of transcription initiation. To determine the relative number of nascent transcripts in each sample, the radiolabeled RNA is purified and hybridized to a membrane containing immobilized DNA from the gene of interest. The amount of radioactivity that hybridizes to the membrane is approximately proportional to the number of nascent transcripts.

Cited by

The DEAD-box helicase Hlc regulates basal transcription and chromatin opening of stress-responsive genes.
Jia R, Lin J, You J, Li S, Shan G, Huang C. Jia R, et al. Nucleic Acids Res. 2022 Aug 11;50(16):9175-89. doi: 10.1093/nar/gkac684. Online ahead of print. Nucleic Acids Res. 2022. PMID: 35950495 Free PMC article.
Transcriptional Run-on: Measuring Nascent Transcription at Specific Genomic Sites in Yeast.
Begley V, de Miguel-Jiménez L, Chávez S. Begley V, et al. Bio Protoc. 2021 Jun 20;11(12):e4064. doi: 10.21769/BioProtoc.4064. eCollection 2021 Jun 20. Bio Protoc. 2021. PMID: 34263006 Free PMC article.
Transcriptional Pausing and Activation at Exons-1 and -2, Respectively, Mediate the MGMT Gene Expression in Human Glioblastoma Cells.
Al-Obaide MAI, Srivenugopal KS. Al-Obaide MAI, et al. Genes (Basel). 2021 Jun 8;12(6):888. doi: 10.3390/genes12060888. Genes (Basel). 2021. PMID: 34201219 Free PMC article.
The long noncoding RNA Synage regulates synapse stability and neuronal function in the cerebellum.
Wang F, Wang Q, Liu B, Mei L, Ma S, Wang S, Wang R, Zhang Y, Niu C, Xiong Z, Zheng Y, Zhang Z, Shi J, Song X. Wang F, et al. Cell Death Differ. 2021 Sep;28(9):2634-2650. doi: 10.1038/s41418-021-00774-3. Epub 2021 Mar 24. Cell Death Differ. 2021. PMID: 33762741 Free PMC article.
Seq'ing identity and function in a repeat-derived noncoding RNA world.
O'Neill RJ. O'Neill RJ. Chromosome Res. 2020 Mar;28(1):111-127. doi: 10.1007/s10577-020-09628-z. Epub 2020 Mar 7. Chromosome Res. 2020. PMID: 32146545 Free PMC article. Review.

MeSH terms

Substances

LinkOut - more resources

Full Text Sources
- HighWire

What is a nuclear run

A nuclear run-on assay was initially developed as a method to establish the transcription rate that plays a part in the regulated expression of mammalian genes. It is used to identify genes that are transcribed at specific times. About a million cell nuclei are isolated, then incubated with labeled nucleotides.

What are nascent transcripts?

Nascent transcripts are retained on the beads in association with the polymerase. The template DNA included a sequence that generates a stem-loop structure in the RNA, close to the 5′ end of the transcript.

What is nascent RNA sequencing?

Nascent RNA sequencing analysis (NRSA) provides a comprehensive view of enhancer-mediated gene regulation. NRSA not only outperforms existing methods for enhancer identification, but also enables annotation and quantification of active enhancers, and prediction of their target genes.

Do all mRNA have poly A tail?

Poly(A) tails are present on almost every eukaryotic mRNA, with the only known exception being some mammalian histone transcripts. Poly(A) tails are added co-transcriptionally and are required for the export of mature mRNAs to the cytoplasm (Fig.