The DuraScribe® T7 and SP6 Transcription Kits produce 2'-Fluorine-modified RNA transcripts called DuraScript® RNA that are completely resistant to RNase A. DuraScript® RNA is more stable in storage and in all RNA applications and can be made and used without the worry and time-consuming procedures normally required for keeping RNA transcripts intact. Recent 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. Double-stranded oligonucleotides, linearized plasmids, or PCR products with a T7 or SP6 promoter can be transcribed in a DuraScribe® transcription reaction.

The DuraScribe® T7 RNA Polymerase, provided in the kits, are mutant forms of T7 RNA Polymerases that efficiently incorporate 2'-Fluorine-CTP (2'-F-dCTP) and 2'-Fluorine-UTP (2'-F-dUTP), as well as ATP and GTP into full length transcripts.(Figure 1) The standard 20-µl DuraScribe® reaction, using 1µg of DNA template, produces approximately 50µg of DuraScript® RNA. The DuraScribe® T7 RNA Polymerase recognizes the same T7 transcription promoters as standard T7 RNA Polymerase.

Figure 1. DuraScribe® T7 and SP6 RNA Polymerases efficiently incorporate 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. The presence of the 2'-F-dC and 2'-F-dU nucleotides render DuraScript RNA completely resistant to the ubiquitous RNase A. DuraScript RNA retains sensitivity to RNase T1 and RNase H. DuraScript RNA is resistant to DNase I degradation so that DuraScribe reactions can be treated to remove the DNA template after the transcription reaction is complete. Double-stranded DuraScript RNA can be digested by RNase III.

	Figure 2. 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. 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.
	Figure 3. 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
	Figure 4. 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.

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Protocols for: DuraScribe® T7 Transcription Kits

Due to the constant updating of the protocols by the manufacturer we have provided a direct link to Lucigen’s product page, where the latest protocol is available.

Please note this will open a new page or window on your computer.

 DuraScribe® Protocol

(catalogue number DS010910 / DS10925 / DS041010)

Please note: all protocols off site are the responsibility of the products supplier

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References

Shi X, Song P, Tao S, Zhang X, Chu CQ. Silencing RORyt in human CD4 T Cells with CD30 aptamer-RORyt shRNA chimera. Sci Rep. 2019;9(1):10375.


Sun, G. and Riggs, A. (2017). Asimple and cost effective approach for in vitro production of sliced siRNAs as potent triggers for RNAi. Molecular therapy - Nucleic acids, 8, pp.345-355


Soldevilla MM, Villanueva H, Casares N, et al. MRP1-CD28 bi-specific oligonucleotide aptamers: target costimulation to drug-resistant melanoma cancer stem cells. Oncotarget. 2016;7(17):23182-23196.


Hervas-Stubbs S, Soldevilla MM, Villanueva H, Mancheno U, Bendandi M, Pastor F. Identifiction of TIM3 2'-fluoro oligonucleotide aptamer by HT-SELEX for Cancer immunotherapy. oncotarget. 2016;7(4):4522-4530


Dua P, Ren S, Lee SW, et al. Cell-SELEX based identifiction of an RNA aptamer for Escherichia coli and its use in various detection formats. Mol cells. 2016;39(11):807-813


Leach, J., Wang, A., Ye, K. and Jin, S. (2016). A RNA-DNA hybird aptamer for Nanoparticle-based prostate Tumor targeted drug delivery. International journal of Molecular Sciences, 17(3),p.380.


Zhen S, Takahahi Y, Narita S, Yang YC, Li X. Targeted delivery of CRISPR/Cas9 to prostate cancer by modified gRNA using a flexible amptamer-cantonic liposome. Oncotarget. 2017;8(6):9375-9387


Kong HY, Byun J. Screening and characterization of a novel RNA aptamer that specifically binds to human prostatic acid phosphatase and human prostate cancer cells. Mol Cells. 2015;38920:171-179.


Stovall GM, Bedenbaugh RS, Singh S, et al. In vitro selection using modified or unnatural nucleotides. Curr protoc nucleic acid chem. 2014;56:9.1-9.6.33. Published 2014 Mar 26


Green, N., Moody, K., Debatis, M. and Marshak-Rothstein, A. (2012). Activation of autoreactive B cells by endogenous TLR7 and TLR3 RNA ligans. Journal of Biological Chemistry, 287(47), pp.39789-39799.


Meis, J.E. and Chen, F. (2002) EPICENTRE Forum 9 (1), 10.

 Capodici, J. et al. (2002) J. Immunol. 169, 5196.

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FAQ: 

AmpliScribe™ T7-Flash™and DuraScribe® T7In VitroTranscription Kits

1. What is the longest in vitro transcript readily made with an AmpliScribe T7-Flash Transcription Kit? 

   While we have tested and readily produced 9-kb transcripts at Epicentre, our customers report high-quality transcripts greater than 11 kb.

2. I’m using less than the recommended 1 µg of DNA template in an AmpliScribe T7-Flash reaction. How can I improve my RNA yield? 

   With lower concentrations of DNA template, you can increase RNA yields by increasing the reaction time from the recommended 30 minutes up to 2-4 hours, depending on the amount of template used. Increasing the reaction temperature from 37°C to 42°C also improves the yield.

3. Why should I set up the AmpliScribe T7-Flash reactions at room temperature? Won’t that affect the RNA polymerase enzyme activity? 

   The components of the reagents in a standard 20-µl reaction are very close to their solubility limits. If you set up the reaction on ice, some reaction components (such as the buffer and the nucleotides) will precipitate. After components precipitate, warming the reaction tube only partially resolubulizes the reagents. The AmpliScribe T7-Flash Enzyme Solution is added last, so it may be kept on ice until needed.

4. How clean does the template DNA need to be for in vitro transcription? 

   Very clean template DNA ensures the best performance of an in vitro transcription reaction. If using a PCR product for the template, purify the desired product from the reaction to remove any remaining primers, primer-dimers, and residual dNTPs. If you are using a plasmid for the template, in order to transcribe the desired RNA product, completely linearize the plasmid, leaving a blunt or 5´-overhanging end. Uncut plasmid serves as excellent template, but the RNA polymerase will transcribe past the desired transcription stop point and can continue around the plasmid several times before the reaction finally stops, creating an RNA that is far longer than desired and includes undesirable vector sequences.

5. What is the shortest in vitro transcript readily made with an AmpliScribe T7-Flash Transcription Kit? 

   We have produced 26-base RNA transcripts with an AmpliScribe T7-Flash Transcription Kit.

6. Can I make nonradioactive, labeled RNA using an AmpliScribe T7-Flash Kit (with labeled nucleotides or by end-labeling)? 

   Yes, you can directly incorporate derivatized nucleotides (with moieties like Cy5, biotin, or digoxygenin) into the transcripts or you can perform post-transcriptional labeling of purified RNA transcripts at the 5´ or 3´ ends. Please contact us for specific protocols.

7. What advantages does an AmpliScribe T7-Flash Transcription Kit have over the standard AmpliScribe High-Yield Transcription Kit? 

   The two main advantages of the AmpliScribe T7-Flash™ Kit are: a) improved RNA yields, even better than the excellent results obtained with the AmpliScribe High-Yield Transcription Kits; and b) a fast, 30-minute procedure.

8. Can I make radiolabeled probes with any of the AmpliScribe High-Yield or T7-Flash transcription kits? 

   Yes, but not recommended. While rarely done any more, radioactive probes may be made using post-transcriptional labeling. Generating radioactive RNA during the in vitro transcription reaction requires a lot of radioactive nucleotide due to the high concentrations of radioactive NTPs required to prepare probes with high specific activity. This is extremely expensive and potentially dangerous. You can prepare radioactive RNA probes by using alkaline phosphatase to generate a 5´-hydroxyl end, followed by radioactive tagging using γ^-32P-ATP and T4 Polynucleotide Kinase, or by using α^-32P-(5´,3´)-bisphosphate NDPs and ligating to the 3´ end of the RNA using T4 RNA Ligase.

9. I am making a template for in vitro transcription using the AmpliScribe Kit. The template structure contains a combined double-stranded T7 promoter and a single-stranded template section. Will AmpliScribe work with this template? 

   While reports of successful hybrid templates for in vitro transcription have been reported, you should use fully-double stranded templates for the best results when transcribing RNA using AmpliScribe and DuraScribe kits.

10. What is the difference between RNA made with the DuraScribe T7 Transcription Kit and RNA made with other in vitro transcription kits? 

    The DuraScribe T7 Transcription Kit produces RNA that contains nucleotides with a 2´ ribose fluorine and that is resistant to degradation by A-type RNases (like the RNase found on human skin), while the AmpliScribe T7-Flash and other transcription kits make standard RNA. DuraScript™ RNA can be reverse-transcribed, like regular RNA, and can be digested by RNase III. However, DuraScript RNA cannot be used as a template to produce proteins by in vitro translation.

11. My AmpliScribe reaction had precipitate while setting up the reaction. Is this normal? 

    AmpliScribe reaction set-up is temperature-sensitive and loss of yield may occur if you use cold reagents when assembling the reaction. All components, including enzymes, should be brought to room temperature to minimize precipitate formation. If precipitates form while setting up your reaction, re-solubilizing may be incomplete. This may impact downstream RNA yield. We suggest running an in vitro transcription reaction using the control template to confirm yields are within kit specification.

12. My AmpliScribe reaction had precipitate in the reaction tube after the protocol’s recommended 30 minutes. Did my reaction fail? 

    Successful AmpliScribe reactions may result in formation of a white precipitate. This precipitate is the synthesized RNA from the reaction – the yields can be so high that the RNA precipitates out of solution. Don't worry – simply dilute the reaction using RNase-free water and purify using any of the recommended methods (ammonium acetate precipitation, spin column, or ethanol precipitation).

13. What is the best procedure for cleanup/DNA template removal to recover the most RNA? 

    It is very simple to clean up AmpliScribe and DuraScribe reactions. We recommend three methods:

    1. Spin column
    2. Salt precipitation (using ammonium acetate with no ethanol for RNA transcripts longer than 100 bases)
    3. Phenol chloroform or ethanol precipitation
14. What is the number of units per microliter of T7 RNA Polymerase in the AmpliScribe T7-Flash Kit?

    Unfortunately, this is considered proprietary. AmpliScribe T7 Polymerase solution contains additives that enhance transcription, so the number of units per AmpliScribe reaction is not comparable to the number of units used per reaction with pure T7 RNA Polymerase.

15. My AmpliScribe reaction had excellent yields, but when I ran it on a native gel the RNA was much shorter than the original template. What could have gone wrong? 

    Single stranded in vitro transcripts are best separated using denaturing gels¹. Denaturing gels allow in vitro transcripts to separate on the basis of their length rather than based on their length secondary structure. When using native gels, sample migration may be altered by secondary structures in the transcripts. 

16. Whenever I use my AmpliScribe Kit I get a really bad smear of RNA during gel electrophoresis instead of a band that is the size of my desired transcript. What is wrong? Did an RNase contaminant get into the reaction? 

No. It is likely that your electrophoresis was performed on a native gel, which does not remove any secondary structures from the RNA. Use denaturing conditions for electrophoresis¹to remove any secondary structures from the RNA and allow the RNA to migrate in a tight band rather than a smear.

1. Molecular Cloning - A Laboratory Manual, Third Edition, 2001. CSHL Press. pp 7.27 - 7.34. J. Sambrook and D. Russell.

* The use of DuraScribe™ T7 Transcription Kit to synthesise nucleic acids with non-canonical bases or for partial ribosubstitution is covered by U.S. patents 5,849,546; 6,107,037 and other patents issued or pending.
These products are accompanied by a limited non-exclusive license for the purchaser to use the purchased product(s) solely for life science research. Contact Epicentre® concerning licenses for other uses.

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Applications

• In situ hybridisation
• Ribonuclease protection assays
• RNA interference (RNAi)
• Competitive RT-PCR assays
• Ribozyme and aptamer studies
• Anti-sense RNA

Benefits

• DuraScript® RNA is completely resistant to RNase A, nucleases found on the human skin
• A standard  reaction produces 50µg of DuraScript® RNA.
• DuraScript® RNA has improved stability in storage and most applications
• No need for gloves
• DuraScribe® T7 RNA Polymerases utilize the same transcription promoters as standard T7 RNA Polymerases.
• Double-stranded DuraScript® RNA is taken up by cultured mammalian cells in the presence of serum and without the need for transfection reagents.
• DuraScript® RNA can be digested by RNase T1 and RNase H, and double-stranded DuraScript® RNA can be digested by RNase III.
• Fluorescently-labelled DuraScribe® RNA can be readily produced.

Figure 5. Forty to sixty µg of a 1.4-kb DuraScript® RNA is produced in a standard 4-hour DuraScribe® T7 Transcription reaction.	Figure 6. A DuraScribe® reaction can be scaled-up to produce even more DuraScript® RNA. Shown is scale up of a DuraScribe T7 Transcription reaction.

* The use of DuraScribe® Transcription Kits to synthesize nucleic acids with non-canonical bases or for partial ribosubstitution is covered by U.S. patents 5,849,546; 6,107,037 and other patents issued or pending. These products are accompanied by a limited on-exclusive license for the purchaser to use the purchased product(s) solely for life science research. Contact EPICENTRE® concerning licenses for other uses.

Note: EPICENTRE® products are licensed under U.S. and international patent rights owned by the Carnegie Institution of Washington
that cover RNA interference. These products are accompanied by a limited non-exclusive worldwide license under the Carnegie Institution of Washington's patent rights for researchers at academic or other not-for-profit institutions to use the product for non-profit research. However, use of dsRNA for RNA interference by for-profit organizations requires a license from the Carnegie Institution
of Washington. For-profit institutions should contact Gloria Brienza of the Carnegie Institution of Washington, 1530 P Street, N.W., Washington, D.C. 20005-1910. E-mail: gbrienza@pst.ciw.edu.

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200