RNA Analysis

In vitro Synthesis & Processing

In vitro synthesis of single-stranded RNA molecules is a routine laboratory procedure that is essential to the rapidly developing field of RNA research. This technique is versatile in that it allows the researcher to tailor synthesis, and introduce modifications that produce a transcript that is optimal for use in downstream applications. Though the ways in which RNA can be produced are countless, the same basic procedure is followed in all synthesis protocols.

The first step in in vitro RNA synthesis is to prepare the DNA template corresponding to the sequence of interest. To allow run off transcription, plasmid DNA template is generally linearized with a restriction enzyme. In addition to plasmid DNA, PCR products and synthetic oligonucleotides can be used as templates for transcription reactions. The template DNA is then transcribed by a T7, T3 or SP6 RNA phage polymerase in the presence of ribonucleoside triphosphates (rNTPs), (1-2). The polymerase traverses the template strand and uses base pairing with the DNA to synthesize a complementary RNA strand (using uracil in the place of thymine). The RNA polymerase travels from the 3' → 5' end of the DNA template strand, to produce an RNA molecule in the 5' → 3' direction (2).


  1. Rio, D. C., et al. RNA: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2011, 205-220.
  2. Cooper, Geoffery M. The Cell: A Molecular Approach. 4th ed. Washington D.C.: ASM Press, 2007. 262-299.
  3. Jani B, Fuchs R. J Vis Exp. 2012 Mar 26 (61). PMID: 22473375
  1. In Vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

    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.

Protocols for In vitro Synthesis & Processing

RNA Reagents Overview

Reagents for RNA Sample Preparation

NEB offers a wide variety of reagents to facilitate sample preparation for downstream applications including next-generation sequencing, library construction and microarrays.

RNA Polymerase Selection Chart

NEB offers RNA polymerases that can be used for in vitro synthesis of RNA for a wide variety of downstream applications. Additional polymerases are offered for the generation of untemplated homoribopolymeric tails for reverse transcription or labeling.

RNA Ligase Selection Chart

NEB offers a variety of ligases for RNA research with unique specificities.

cDNA Synthesis Selection Chart

NEB offers several reagents for cDNA synthesis for use in applications including qPCR and qRT-PCR. For your convenience, reagents are available as kits or standalone products, depending on your needs.

DNA Ligase Selection Chart

While more than one ligase may work for your application, the DNA Ligase Selection Chart present our recommendations for optimal performance.