Modifications to the basic synthesis protocol can facilitate the incorporation of modified nucleotides to obtain biotin-labeled, dye-labeled or high specific activity radiolabeled RNA probes (1).
mRNAs synthesized in vivo by eukaryotic organisms are cotranscriptionally modified by the addition of a a cap structure, which consists of a 5' 7-methyl guanosine residue that protects the transcript from nuclease digestion and promotes translation. Additionally, eukaryotic mRNAs are 3’-polyadenylated by a template independent polymerase. The poly(A) tail also functions in mRNA stability and translational regulation.
For experiments where RNA will be introduced into cells by transfection or microinjection, in vitro transcripts can be modified to mimic mRNA by using the mRNA capping enzymes from vaccinia virus (NEB #M2080) to add cap structures, and E. coli poly(A) polymerase (NEB #M0276) to add poly(A) tails. Alternatively, cap structure analogs, such as (m7G(5')ppp(5')G) (NEB #S1404), may be used to co-transcriptionally cap RNA transcripts during synthesis.
Synthesized RNA can be used in a number of applications including RNA structure and function studies, ribozyme biochemistry, probes for RNase protection assays and hybridization based blots, anti-sense RNA and RNAi experiments, microarray analysis, microinjection, in vitro translation, and RNA vaccines.
- Edited by R. K. Hartman, A. Bindereif, A. Schön, E. Westhof. Handbook of RNA
Biochemistry: Student Edition. Weinheim: WILEY-VCH Verlag GmbH & Co, 2009,
- Jani B, Fuchs R. J Vis Exp. 2012 Mar 26 (61). PMID: 22473375