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  • Title: In vitro transcription and capping of Gaussia luciferase mRNA followed by HeLa cell transfection.
    Author: Jani B, Fuchs R.
    Journal: J Vis Exp; 2012 Mar 26; (61):. PubMed ID: 22473375.
    Abstract:
    In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg(2+) ions. Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing(1), RNAi experiments in mammalian cells(2), antisense RNA amplification by the "Eberwine method"(3), microarray analysis(4) and for RNA vaccine studies(5). The technique can also be used for producing radiolabeled and dye labeled probes(6). Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA(7). The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 μg RNA per 20 μl reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed. Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability(8), efficient translation(9), nuclear transport(10) and splicing(11). The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme(12,13). The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications(14) such as generating viral genomic RNA for reverse-genetic systems(15) and crystallographic studies of cap binding proteins such as eIF4E(16). In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.
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