Search research articles

ABOUT JoVE

Overview Leadership Blog JoVE Help Center

AUTHORS

Publishing Process Editorial Board Scope & Policies Peer Review FAQ Submit

LIBRARIANS

Testimonials Subscriptions Access Resources Library Advisory Board FAQ

RESEARCH

JoVE Journal Methods Collections JoVE Encyclopedia of Experiments Archive

EDUCATION

JoVE Core JoVE Business JoVE Science Education JoVE Lab Manual Faculty Resource Center Faculty Site

Terms & Conditions of Use

Related Concept Videos

RACE - Rapid Amplification of cDNA Ends

RACE - Rapid Amplification of cDNA Ends

Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific primer.
Since the...

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by

Same author

An expanding cityscape and its multi-scale effects on lizard distribution.

Frontiers in conservation science·2024

Same author

Animal movement ecology in India: insights from 2011-2021 and prospective for the future.

PeerJ·2022

Same author

Acoustically eavesdropping bat predators take longer to capture katydid prey signalling in aggregation.

The Journal of experimental biology·2021

Same journal

CODEC Library Preparation From Genomic DNA.

Current protocols·2026

Same journal

Standardized Pipeline for Metabolism and Cognition in GO-DS21 Mouse Model: Investigating Down Syndrome Comorbidities.

Current protocols·2026

Same journal

Effect of Passive Hamstring Stretching on Craniovertebral Angle, Muscle Activation Pattern and Proprioception in Individuals with Forward Head Posture: Study Protocol for Randomized Controlled Trial.

Current protocols·2026

Same journal

Standardization of Immunohistochemistry Using an Anti-WASLB Antibody in the Skeletal Muscle of Teleost Fish.

Current protocols·2026

Same journal

Zebrafish Embryos to Profile Nano(bio)materials: A Modular Platform for Developmental Toxicity, Neurotoxicity, and Inflammation-Regeneration Assays.

Current protocols·2026

Same journal

Differentiation of 3T3-L1 Fibroblasts Into Adipocytes.

Current protocols·2026

See all related articles

Search research articles

Home
Streamlined In Vitro Transcription For Generating Self-amplifying Rna With Modified Nucleotides.

Home
Streamlined In Vitro Transcription For Generating Self-amplifying Rna With Modified Nucleotides.

Related Experiment Video

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

Published on: November 13, 2014

Streamlined In Vitro Transcription for Generating Self-Amplifying RNA With Modified Nucleotides.

Harish Prakash^1,2, Karthik Kadhir Velu^1,2, Vasanth Thamodaran^1,3

¹Tata Institute for Genetics and Society, Bengaluru, India.

Current Protocols

|June 19, 2026

View abstract on PubMed

Summary

This summary is machine-generated.

This study presents a streamlined protocol for producing modified self-amplifying RNA (saRNA) using Venezuelan equine encephalitis virus (VEEV) backbones. The optimized method enhances RNA amplification and protein expression for advanced biological applications.

Keywords:

RNA purification chemically modified nucleotides in vitro transcription self‐amplifying RNA transfection

More Related Videos

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

Published on: December 29, 2021

Reverse Genetics to Engineer Positive-Sense RNA Virus Variants

Reverse Genetics to Engineer Positive-Sense RNA Virus Variants

Published on: June 9, 2022

Related Experiment Videos

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

Published on: November 13, 2014

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

Published on: December 29, 2021

Reverse Genetics to Engineer Positive-Sense RNA Virus Variants

Reverse Genetics to Engineer Positive-Sense RNA Virus Variants

Published on: June 9, 2022

Area of Science:

Molecular Biology
RNA Therapeutics
Virology

Background:

Self-amplifying RNA (saRNA) offers advantages over mRNA for protein expression.
Venezuelan equine encephalitis virus (VEEV)-based saRNA is efficient but requires optimization for modified nucleotides.
Chemical modifications like N1-methylpseudouridine can impede saRNA amplification.

Purpose of the Study:

To develop a streamlined protocol for generating 5-methylcytidine (m5C)-modified VEEV-based saRNA.
To optimize saRNA production for enhanced intracellular amplification and protein expression.
To provide a reproducible method for creating chemically modified saRNA for research.

Main Methods:

Utilized a single-step in vitro transcription (IVT) strategy.

Employed PCR-generated DNA templates with encoded poly(A) tails.

Incorporated CleanCap AU for co-transcriptional capping and post-transcriptional phosphatase treatment.

Purified saRNA using the phenol-chloroform-isoamyl alcohol method.

Main Results:

Successfully generated m5C-modified VEEV-based saRNA with enhanced functionality.
Demonstrated reduced innate immune activation and improved expression durability.
Validated the protocol through functional assessment in HEK293T cells.

Conclusions:

The developed protocol provides a reproducible framework for producing capped, tailed, and chemically modified saRNA.
This method is suitable for downstream functional and translational studies.
Optimized saRNA production holds promise for advancing RNA-based therapeutics and research.