Thesis
Developing a nuclear run-on assay to study transcription-genome organization coupling
Master of Science (M.S.), Drexel University
Jul 2026
DOI:
https://doi.org/10.17918/00011510
Abstract
Three-dimensional genome organization is closely associated with transcriptional regulation, but conventional steady-state RNA measurements do not directly report the activity of transcriptionally engaged RNA polymerases. Nuclear run-on assays address this limitation by allowing polymerases retained in isolated nuclei to extend nascent transcripts under defined reaction conditions. The objective of this study was to develop and evaluate a semi-in vitro nuclear run-on assay for measuring nucleotide-dependent RNA extension in isolated HCT-116 nuclei. Nuclei were divided between an all-NTP condition containing ATP, CTP, GTP, and UTP and a GTP-only background-control condition. A defined GFP RNA spike-in was added before RNA purification to normalize technical variation. Assay performance was assessed by RT-qPCR and RNA sequencing. RT-qPCR showed a larger HPRT1 Exon 1 signal in the all-NTP condition than in the GTP-only condition across three technical wells from a single experiment (exploratory technical-well comparison, P < 0.05). Because no independent biological replicates were performed, this result is interpreted as preliminary rather than as evidence of biological reproducibility. GFP remained stable, as expected for the normalization control, and GAPDH did not differ between conditions. Primer sets targeting later HPRT1 and GAPDH exon positions produced weak, inconsistent, or undetectable signal. RNA sequencing provided descriptive genome-wide support for nucleotide-dependent RNA extension. More genes showed increased than decreased signal in the all-NTP condition at both fold-change thresholds examined. Most differences were modest, and no Gene Ontology Biological Process term remained significant after multiple-testing correction, consistent with a broad assay effect rather than activation of a specific biological program. Together, these findings support the feasibility of a proof-of-principle semi-in vitro nuclear run-on workflow for detecting nucleotide-dependent RNA extension in isolated HCT-116 nuclei. The study was limited to assay development and initial evaluation. Selective enrichment of newly extended RNA, independent biological replication, and broader positional validation will be required to improve assay specificity, reproducibility, and quantitative performance.
Metrics
1 Record Views
Details
- Title
- Developing a nuclear run-on assay to study transcription-genome organization coupling
- Creators
- Christopher Miranda
- Contributors
- Yu Liu (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Master of Science (M.S.)
- Publisher
- Drexel University
- Number of pages
- vi, 38 pages
- Resource Type
- Thesis
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology; College of Medicine; Drexel University
- Other Identifier
- 991022196749804721