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).
- Rio, D. C., et al. RNA: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor
Laboratory Press, 2011, 205-220.
- Cooper, Geoffery M. The Cell: A Molecular Approach. 4th ed. Washington D.C.: ASM Press, 2007. 262-299.
- Jani B, Fuchs R. J Vis Exp. 2012 Mar 26 (61). PMID: 22473375