In Vitro Rna
In Vitro Rna - Related Videos
10:34
Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture
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Cited by 19 •
2016
An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.
10:52
Visualization and Quantification of Intermolecular RNA Base Pairing in in vitro RNA Clusters Using Split Broccoli RNA Reporters
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2026
This protocol describes a visual assay using split Broccoli RNA reporters to detect and quantify intermolecular base pairing in RNA clusters in vitro.
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.
08:55
In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection
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Cited by 13 •
2012
This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.
09:16
Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro
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Cited by 1 •
2014
RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.
FAQs
During cell-free transcription, a DNA template supplies the sequence information, while an RNA polymerase builds an RNA strand using nucleotide triphosphates. Salts and other reaction conditions support the activity of this system. Because these components are supplied in a defined mixture, researchers can examine how a chosen template and reaction environment affect the RNA produced.
Controlled conditions make it possible to vary one part of an RNA experiment while holding other factors stable. Researchers can adjust the supplied template, nucleotide triphosphates, salts, or reaction conditions and then study the resulting RNA. This control helps connect observed differences in RNA behavior to experimental variables rather than to changes occurring inside a living cell.
Cell-free systems separate RNA production or analysis from the many processes present in living cells. This allows researchers to focus on RNA structure, function, processing, or molecular interactions under selected laboratory conditions. In contrast, cellular studies examine RNA within its biological environment. Using the two approaches can provide complementary information about RNA behavior and post-transcriptional regulation.
A basic setup uses a DNA template, an RNA polymerase, nucleotide triphosphates, salts, and appropriate reaction conditions. The polymerase copies the template in the supplied mixture, producing the RNA that will be examined or used in a later study. This component-based design lets investigators define the sequence source and chemical environment rather than relying on a cell.
Researchers can use these preparations to examine RNA structure, function, processing, and interactions. Each focus addresses a different aspect of RNA biology: structure concerns molecular form, function concerns biological activity, processing concerns changes to the RNA, and interactions concern relationships with other molecules. The approach therefore supports mechanistic studies and analysis of post-transcriptional regulation.
Cell-free production provides a way to obtain messenger RNA for gene-expression studies and therapeutic development while keeping the RNA sequence and experimental conditions under precise control. The same controlled approach supports vaccine research. In biology, this is valuable because investigators can connect a defined RNA design with its experimental use without introducing the additional variables of a living-cell production system.