In Vitro Transcription
In Vitro Transcription - Related Videos
09:28
In Vitro Transcription Assays and Their Application in Drug Discovery
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Cited by 6 •
2016
In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.
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.
02:16
Transcription Factors
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2019
Tissue-specific transcription factors contribute to diverse cellular functions in mammals. For example, the gene for beta globin, a major component of hemoglobin, is present in all cells of the body. However, it is only expressed in red blood cells because the transcription factors that can bind to the promoter sequences of the beta globin gene are only expressed in these cells. Tissue-specific transcription factors also ensure that mutations in these factors may impair only the function of...
02:23
Master Transcription Regulators
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2020
Master transcription regulators are regulatory proteins that are predominantly responsible for regulating the expression of multiple genes. Often these genes work in concert to drive a complex process. Activation of a master transcription regulator can lead to a cascade of transcriptional activation necessary for that outcome. These regulators can directly bind to the regulatory sequences of the various genes involved, or they can indirectly regulate transcription by binding to regulatory...
02:35
Transcription Elongation Factors
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2020
Transcription elongation is a dynamic process that alters depending upon the sequence heterogeneity of the DNA being transcribed. Hence, it is not surprising that the elongation complex's composition also varies along the way while transcribing a gene. The transcription elongation is regulated via pausing of RNA polymerase on several occasions during transcription. In bacteria, these halts are necessary because the transcription of DNA into mRNA is coupled to the translation of that mRNA into a...
FAQs
The promoter provides the recognition site that directs RNA polymerase to the DNA template. Once positioned, the enzyme incorporates complementary ribonucleotides, extending an RNA transcript according to the template sequence. This sequence-directed mechanism allows investigators to generate defined RNA molecules rather than relying on transcription occurring within a cell.
Temperature, ion availability, and ribonucleotide concentrations can change both transcript yield and RNA quality. These variables influence how efficiently RNA polymerase uses the DNA template and incorporates substrates. Researchers therefore adjust the reaction environment when seeking sufficient amounts of a transcript while maintaining the desired quality for downstream biological studies.
Because the reaction occurs outside living cells, investigators can produce defined RNA molecules under controlled conditions. This format separates transcript production from cellular processes and provides a system for examining RNA synthesis directly. Such control is useful when researchers need specific messenger RNAs, guide RNAs, riboprobes, or other experimentally defined transcripts.
A typical workflow begins with a DNA template containing a promoter recognized by the selected RNA polymerase. The template is combined with ribonucleotides and the reaction environment is established by controlling temperature and ion conditions. The polymerase then generates the RNA transcript, with yield and quality serving as important outcomes of the procedure.
The method can produce messenger RNA, riboprobes, guide RNAs, and other defined transcripts. These products support gene expression studies, molecular assays, and protein production. Because researchers select the DNA template and reaction conditions, they can generate RNA suited to a particular experimental question instead of using a single transcript type for every application.
Controlled RNA production enables investigations of RNA structure, function, and processing without requiring the transcript to be generated inside living cells. The resulting molecules also contribute to therapeutic development. In biology, this makes the technique useful both for studying fundamental RNA behavior and for producing defined materials for applied research.