What is In-Vitro Transcribed RNA? A Powerful Tool for Molecular Biology
In-vitro transcribed (IVT) RNA is a laboratory-generated RNA molecule synthesized from a DNA template outside of living cells. This versatile technique allows scientists to produce customized RNA for use in research, diagnostics, and even therapeutics—such as the mRNA vaccines that made headlines during the COVID-19 pandemic.
If you're a student, researcher, or biotech enthusiast curious about how IVT RNA is made and used, this post is for you.
What is In-Vitro Transcription?
In-vitro transcription is the process of generating RNA from a DNA template using an RNA polymerase enzyme, such as T7, SP6, or T3. The DNA template must contain a promoter sequence that the enzyme recognizes and binds to, enabling the RNA synthesis process.
Unlike cellular transcription, in-vitro transcription is done in a controlled lab environment, giving scientists full control over the RNA product—its sequence, structure, modifications, and length.
What Do You Need for an In-Vitro Transcription Reaction?
A standard IVT RNA reaction includes:
DNA Template
Either a linearized plasmid or a PCR-amplified DNA fragment with a promoter (T7, SP6, or T3).RNA Polymerase
Enzyme specific to the promoter used. T7 is the most commonly used polymerase.NTPs (Nucleoside Triphosphates)
ATP, CTP, GTP, and UTP are the building blocks for RNA. These can also be chemically modified depending on the application.Buffer
Contains salts, pH stabilizers, and Mg²⁺ to support enzymatic activity.RNase Inhibitor
Protects RNA from degradation by RNases during the reaction.
Optional components include:
Capping enzyme or cap analog
Essential for mRNA stability and translation in eukaryotic systems.Poly(A) polymerase
Adds a poly-A tail to enhance RNA stability and translation efficiency.Modified nucleotides
Such as pseudouridine or 5-methylcytidine, commonly used to reduce immunogenicity in therapeutic RNAs.
What is In-Vitro Transcribed RNA Used For?
IVT RNA has a broad range of applications:
mRNA Expression Studies
Introduce mRNA into cells or embryos to transiently express proteins.Therapeutic Development
mRNA vaccines and RNA therapeutics rely on IVT RNA technology.RNA Probes
For Northern blotting or in situ hybridization to detect gene expression.Standard Curves in qPCR/ddPCR
Create precise RNA standards for absolute quantification.Guide RNAs (gRNAs) for CRISPR
Generate short RNA sequences that guide Cas9 to DNA targets.
Tips for Success with IVT RNA
Linearize plasmids before transcription to prevent run-off transcripts.
Always use RNase-free tools and reagents.
Purify your RNA using column-based kits or lithium chloride precipitation to remove enzymes, salts, and DNA.
Run a small aliquot on a denaturing agarose gel or Bioanalyzer to check RNA integrity.
Store at –80°C in small aliquots to avoid freeze-thaw degradation.
Kits and Reagents for IVT RNA
Some popular kits and suppliers include:
Thermo Fisher Scientific: MEGAscript™ and mMESSAGE mMACHINE™ Kits
New England Biolabs (NEB): HiScribe™ T7 Quick and High Yield RNA Synthesis Kits
Promega: RiboMAX™ Large Scale RNA Production Systems
TriLink BioTechnologies: CleanCap® capping technology for mRNA production
Conclusion
In-vitro transcribed RNA is a cornerstone of modern molecular biology and biotechnology. Whether you’re producing mRNA for transfection, designing CRISPR experiments, or creating RNA standards, IVT RNA offers flexibility, precision, and scalability. Mastering this technique opens the door to cutting-edge applications in research and therapeutic development.
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