Newly Transcribed Rna Purification
Newly Transcribed Rna Purification - Related Videos
11:00
Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture
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Cited by 105 •
2013
Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.
03:36
Recovery of Zika Virus from In Vitro-Transcribed RNA
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2026
Source: Weger-Lucarelli, J., et al. Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone. J. Vis. Exp. (2017)This video demonstrates the recovery of infectious Zika virus using electroporation of in vitro transcribed RNA. The process shows how synthetic RNA initiates infection and leads to viral replication in cultured cells.
11:09
Chromatin Isolation by RNA Purification (ChIRP)
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Cited by 380 •
2012
ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.
02:30
Isolation of Small Regulatory RNA–Bound Bacterial Target RNA Using Affinity Purification
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2025
Source: Mercier, N., et. al., MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria. J. Vis. Exp. (2021)This video demonstrates the isolation of MS2-tagged small regulatory RNA (sRNA) bound to its bacterial target RNA using affinity purification with a maltose-binding protein (MBP)–MS2 coat protein fusion immobilized on amylose resin.
08:29
Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA
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Cited by 6 •
2019
Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.
FAQs
Newly transcribed RNA purification uses metabolic labeling to mark transcripts made during a selected interval. A modified nucleotide such as 4-thiouridine becomes part of those RNA molecules, allowing them to be distinguished from unlabeled preexisting RNA. Chemical tagging then creates the affinity handle needed for selective capture and downstream analysis.
The labeling interval determines which transcriptional events the isolated RNA represents. A defined window can capture an early response or a later phase of gene regulation, while changes in the window alter the biological time point being measured. This timing makes it possible to examine RNA synthesis and processing as dynamic processes rather than static cellular states.
Total RNA contains both older molecules and recently produced transcripts, so its abundance may change slowly or obscure an immediate transcriptional response. Separating the newly made fraction provides a more direct view of current RNA synthesis and emerging processing changes. This distinction helps researchers interpret whether gene-expression differences reflect active regulation rather than accumulated RNA.
After labeled RNA is chemically modified, biotin can serve as the affinity tag used for isolation. Streptavidin recognizes and binds biotin, enabling the tagged RNA fraction to be captured separately from untagged RNA. This pairing supplies the molecular connection between the labeling step and physical enrichment of transcripts synthesized during the selected interval.
The workflow begins by exposing cells to a modified nucleotide during a defined labeling period. RNA is then isolated, the labeled transcripts are chemically tagged, and the tagged fraction is captured through an affinity interaction, often involving biotin and streptavidin. The purified newly made RNA can subsequently be examined for transcriptional responses, synthesis, or processing changes.
The captured fraction represents RNA associated with the labeling period, whereas the unselected pool contains RNA that was not captured through that labeling-dependent strategy. Interpretation therefore depends on the defined time window and the separation between labeled and preexisting molecules. Comparing these fractions can clarify how gene regulation changes over time.
It is useful when the research question concerns how quickly cells alter RNA production after a biological change, rather than only the final abundance of cellular RNA. The approach supports analysis of transcriptional responses, RNA synthesis, and RNA processing over time. It can therefore add temporal resolution to gene-expression studies in biology and disease-related research.
Disease-related studies can use the newly produced RNA fraction to examine whether altered gene expression reflects ongoing transcriptional regulation or differences in the preexisting RNA pool. By focusing on transcripts generated during a defined interval, researchers can investigate temporal changes in cellular responses and RNA processing that may be obscured in measurements of total RNA.