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In Vitro Transcription
In Vitro Transcription Kits
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AmpliScribeTM & DuraScribe

In Vitro Transcription Kit

AmpliScribe™ T7-Flash™ Transcription Kit

Applications
  • Rapid, maximum-yield synthesis of RNA for studies of RNA structure, processing, ribozymes, RNAi, or antisense RNA.

The AmpliScribe™ T7-Flash™ Transcription Kit uses Epicentre's rapid, high-yield in vitro transcription technology. An AmpliScribe T7-FlashTranscription reaction yields more RNA (180 µg per 20-µl reaction) in 30 minutes than other high-yield in vitro transcription kit reactions produce in 2 hours (Fig. 1), including Epicentre's own AmpliScribe T7 High Yield Kit. The AmpliScribe T7-Flash Kit utilizes proprietary enzyme formulations that enable the maximum possible yields of RNA from virtually any template that is downstream of the T7 RNA polymerase promoter, including sequences in linearized plasmids, cDNA,double-stranded oligos, or PCR products.

Benefits
  • Exceptionally high yields of full-length RNA. Reactions can be scaled up to obtain grams (or more) of RNA.
  • When transcribing 1 µg of 1-kb linearized plasmid DNA template, a standard AmpliScribe T7-Flash reaction is complete in 30 minutes and yields up to 180 µg of RNA.
  • RNase inhibitor included.
  • Can be used for "Eberwine"-type RNA amplification procedures. However, we recommend TargetAmp™ 2-Round aRNA Amplification Kits for amplification of mRNA from laser-capture microdissection or other small samples.

i_ampliscribet7_transreaction.gif
Figure 1. An AmpliScribe™ T7-Flash™ transcription reaction is complete in 30 minutes and yields more RNA than other 2-hour "high-yield" transcription reactions.Yields shown were obtained using 1 µg of a linear DNA template in a 20-µl reaction, producing a 1.4-kb RNA.

Notes:
  1. For high-yield transcription of templates having an SP6 promoter, use the AmpliScribe SP6 High Yield Transcription Kit.
  2. To prepare transcripts that are completely resistant to RNase A, use the DuraScribe® T7 or SP6 Transcription Kits.
  3. Use AmpliCap™ High Yield Message Maker Kits for efficient production of RNA with an m
  4. 7G[5´]ppp[5´]G cap structure on the 5´ end.
  5. Because of the very high concentration of NTPs used in AmpliScribe reactions, this kit is not recommended for preparing high specific-activity radiolabeled RNA probes.


AmpliScribe™ T7-Flash™ Biotin-RNA Transcription Kit

Applications
  • Production of nonradioactive RNA probes for any purpose, including microarray (transcription profiling) studies, in situhybridization, and blotting experiments.

The AmpliScribe™ T7-Flash™ Biotin-RNA Transcription Kit enables high-yield synthesis of biotin-RNA or biotin-antisense RNA (aRNA, also called cRNA). The kit incorporates Biotin-16-UTP directly into the RNA transcript. High yields of biotin-RNA are produced from as little as 50 ng of input DNA template.


Benefits
  • The kit uses Epicentre's AmpliScribe T7-Flash rapid, high-yield in vitro transcription technology to generate the highest possible yield of biotin-RNA.
  • In vitro
  • transcription reaction conditions are optimized to yield optimal levels of uniformly labeled biotin-RNA for high signal intensity.
  • RNase Inhibitor included.

스크린샷 2013-06-17 오후 1.03.08.png
Table 1. Yield of Biotin-RNA (in mg) from varying amounts of control template DNA from a standard 37°C, 20 ml AmpliScribe T7-Flash Biotin-RNA Transcription Kit reaction over time. Results may vary depending on the template used.



AmpliScribe™ T7 High Yield Transcription Kit

Applications
  • High-yield synthesis of RNA from DNA cloned downstream from a T7 RNA polymerase promoter, for microinjection, RNA interference, or antisense experiments.
  • Structural and functional studies of splicing, processing, heterogeneous nuclear RNAs, tRNAs, viral RNAs, and ribozymes.1
  • Production of fluorescent2-, biotinylated3-, or digoxigenin4- labeled RNA.

The AmpliScribe™ T7 High Yield Transcription Kit is specially formulated to use high concentrations of NTPs, that can be inhibitory to other in vitro transcription systems. AmpliScribe High Yield Transcription reactions can produce >20-fold more full-length RNA transcript than conventional in vitro transcription reactions. For example, the AmpliScribe T7 Kit yields approximately 5-7.5 mg/ml of 1.4-kb full-length control RNA in 30-120 minutes, respectively, using 1 µg of template DNA (Fig. 1).


Benefits
  • High yields of RNA from all templates—synthesize both long (Fig. 2) and short RNA using only one kit.
  • 5 Produce microgram amounts of RNA from as little as 1 ng of template.
  • Reactions can be scaled up to produce milligram amounts of RNA.
  • Produce >20 times more RNA than conventional or "homemade" in vitro transcription reactions.
  • RNase inhibitor included.

Note
: Because of the very high concentration of NTPs used in AmpliScribe reactions, these kits are not recommended for preparing high specific-activity radiolabeled RNA probes.


References
  1. Johnson, M. (1995) Epicentre Forum 2(5), 4.
  2. DeLong, E.F. et al. (1999) App. and Environ. Microbiol. 65, 5554.
  3. Kaplan, E. et al. (1996) Epicentre Forum 3(2), 1.
  4. Hoff man, L.M. and Johnson, M.G. (1994) BioTechniques 17, 372.
  5. Schanke, J. (2000) Epicentre Forum 7(2), 6.

i_ampliscribet7_transkit.gif
Figure 1. The AmpliScribe™ T7 High Yield Transcription Kit is specially formulated to consistently produce >20-fold more RNA than a conventional T7 in vitro transcription reaction.
g_ampliscribet7_fulllengthrna.jpg
Figure 2. An AmpliScribe™ T7 High Yield Transcription reaction produces full-length, long RNA transcripts. Aliquots from standard 20-µl AmpliScribe T7 High Yield Transcription reactions were loaded onto a 1% agarose-formaldehyde gel and stained with ethidium bromide. The size of the RNA transcripts and RNA yields (in parentheses) were: Lane 1, 3.0 kb (185 µg); lane 2, 4.0 kb (178 µg); lane 3, 5.0 kb (176 µg); lane 4, 6.0 kb (169 µg); lane M, RNA size marker.





DuraScribe® T7 Transcription Kit

Applications
  • High-yield synthesis of RNase A-resistant RNA for in situhybridization, ribonuclease protection assays, RNA interference (RNAi),
  • 1 ribozyme studies, antisense RNA studies, and RNA aptamer preparation.

The DuraScribe® T7 Transcription Kit* produces 2´-fluorine-modified RNA transcripts—called DuraScript® RNA—that are completely resistant to RNase A (Fig. 1).
1 DuraScript RNA is more stable in storage and in all RNA applications, and can be made and used without the tedious procedures normally required for keeping RNA transcripts intact. Research indicates that double-stranded DuraScript RNA can be delivered into cultured mammalian cells in the presence of serum and without the need for transfection reagents.2 Double-stranded oligonucleotides, linearized plasmids, or PCR products with a T7 promoter can be transcribed in a DuraScribe transcription reaction.

The DuraScribe T7 RNA Polymerase provided in the kit efficiently incorporates 2´-Fluorine-CTP (2´-F-dCTP) and 2´-Fluorine-UTP (2´-F-dUTP), as well as ATP and GTP into full length transcripts. The standard 20-µl DuraScribe reaction, using 1 µg of DNA template, produces approximately 50 µg of DuraScript RNA (Fig. 2). The DuraScribe T7 RNA Polymerase recognizes the same T7 transcription promoter as the standard T7 RNA Polymerase.

Benefits
  • DuraScript RNA is completely resistant to RNase A and to the nucleases found on human skin.
  • No need for gloves, DEPC-treatment of reagents, or RNase inhibitors when making or working with DuraScript RNA.
  • Double-stranded DuraScript RNA is taken up by cultured mammalian cells in the presence of serum and without the need for transfection reagents.2
  • Fluorescently labeled DuraScribe RNA can be readily produced.

g_durascribe_rnaseadigestion.jpg
Figure 1. DuraScript® RNA is resistant to RNase A digestion. A 1.4-kb standard RNA transcript and a 1.4-kb DuraScript RNA transcript were each incubated with 1 U of highly purified RNase A for 30 minutes. The standard RNA transcript was completely degraded while the DuraScript RNA transcript remained intact. Lane M, size ladder; lane 1, 1.4-kb standard RNA transcript; lane 2, standard RNA after RNase A treatment; lane 3, 1.4-kb DuraScript RNA; lane 4, DuraScript RNA after RNase A treatment.

i_durascribe_rnayield.gif
Figure 2. Yield of RNA from a DuraScribe® T7 Transcription reaction. A standard reaction (4-6 hours) produced 40-60 µg of a 1.4-kb DuraScript® RNA.

i_durascribe_full_length.gif
Figure 3 (click to enlarge). The DuraScribe® T7 RNA Polymerase efficiently incorporates 2´-F-dCTP and 2´-F-dUTP into full length DuraScript® RNA. The presence of the fluorine at the 2´-position of the 2´-F-dC and 2´-F-dU nucleotides prevents RNase A digestion. The result is DuraScript® RNA that is completely resistant to RNase A and related ribonucleases.

gel_fig2_new2_copy.jpg









Figure 4. DuraScript® RNA is completely resistant to "finger" nucleases. A 1.4-kb standard RNA transcript and a 1.4-kb DuraScript RNA transcript were produced using sterile water or water that had been contaminated by exposure to the hands of a test subject. The standard RNA shows extensive degradation from "finger" nucleases in the contaminated water while the DuraScript RNA remains fully intact. M, Size ladder; Lane 1, Standard RNA transcript; Lane 2, Standard RNA after "finger" nuclease exposure; Lane 3, DuraScript RNA; Lane 4, DuraScript RNA after "finger" nuclease exposure.






durascript_fig3.gif 









Figure 5. DuraScript® RNA is stable in tissue culture media for at least 2 hours. Five micrograms of a 1.4-kb standard RNA transcript and a DuraScript RNA transcript were incubated in 100 µl of tissue culture media (D-MEM + 10% fetal calf serum) at 37°C. Lane 1, RNA ladder; Lane 2, standard RNA; Lane 3, standard RNA after 15 minutes in tissue culture media; Lane 4, DuraScript RNA; Lanes 5-8 DuraScript RNA after 15, 30, 60, and 120 minutes, respectively, in tissue culture media.


References
  1. Capodici, J. et al. (2002) J. Immunol. 169, 5196.
  2. Meis, J. and Chen, F. (2002) Epicentre Forum 9(1), 10.
* Covered by issued and/or pending patents.
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