Capped RNA that Gives Higher Protein Yields mMESSAGE mMACHINE™ T7 Ultra
The mMESSAGE mMACHINE T7 Ultra Kit produces high quality, efficiently capped in vitro transcribed mRNA that translates with much greater efficiency than RNA synthesized with conventional cap analog. Ensuring that cap is added to RNA transcripts only in the correct orientation increases translation efficiency during both in vitro and in vivo translation experiments. Ambion's Poly(A) Tailing Kit is incorporated into the mMESSAGE mMACHINE T7 Ultra Kit for production of stable RNA specifically for microinjection and transfection.
ARCA-capped RNA is Translationally More Active
Figure 1. Schematic of ARCA Molecule.
Figure 2. Transcripts Containing ARCA Are More Highly Translated In Transfected Cells. Comparison of protein expression from standard and ARCA capped luciferase RNAs, plus and minus poly(A) tail, at different time points after transfection. A. Either standard cap analog-capped or ARCA-capped luciferase in vitro transcribed RNA (1 µg) was transfected into HeLa cells. Cells were harvested and lysed at 8 hr, 10 hr, 12 hr, 24 hr and 48 hr post-transfection. Luciferase activity was measured and plotted against transfection time. B. Poly(A)-tailed luciferase RNA (1 µg) prepared in either a standard mMESSAGE mMACHINE™ or a mMESSAGE mMACHINE Ultra reaction was transfected as above. Luciferase activity was measured and plotted against time following transfection.
Figure 3. Transcripts Containing ARCA Are More Highly Translated In Microinjected Embryos. Comparison of protein expression, from standard Cap Analog and ARCA capped luciferase RNA at different time points after microinjection. Either standard cap analog-capped or ARCA-capped luciferase RNA (100 pg) was microinjected into Xenopus embryos. Embryos were harvested and lysed at 8 hr, 24 hr, 50 hr, and 70 hr post injection. Luciferase activity was measured and plotted against time since microinjection.
The mMESSAGE mMACHINE T7 Ultra Kit combines ARCA and reagents for poly(A) tailing with Ambion's patented high yield transcription technology. The incorporation of ARCA into RNA transcripts is efficient and does not compromise RNA yield (see below). In addition, RNA transcribed with the mMESSAGE mMACHINE T7 Ultra Kit is more stable and more efficiently translated than RNA synthesized by traditional transcription kits. mMESSAGE mMACHINE T7 Ultra transcripts are ideal for both in vitro and in vivo translation experiments.
Capping RNA Transcripts with ARCA
Figure 4. Ratio of Cap Analog:GTP to Maximize Yield of ARCA Capped Transcripts. ARCA was incorporated along with GTP during in vitro transcription system using short (6 nt) and long (1.8 kb) transcripts containing a "G" as the first nucleotide. Cap analogs such as ARCA compete with GTP for incorporation into the first nucleotide position. As the ratio of the cap analog to GTP increases in the reaction, the ratio of capped RNA to uncapped RNA increases proportionally. The short RNA transcripts were resolved on a 20% denaturing polyacrylamide/8 M urea gel and synthesized products were detected by autoradiography.