DuraScribe® SP6 Transcription Kit

Applications

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

The DuraScribe® T7 and SP6 Transcription Kits* produce 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 or SP6 promoter can be transcribed in a DuraScribe transcription reaction.

The DuraScribe T7 and SP6 RNA Polymerases provided in the kits 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. The standard 20-µl DuraScribe reaction, using 1 µg of DNA template, produces approximately 50 µg of DuraScript RNA (Fig. 2). The DuraScribe T7 and SP6 RNA Polymerases recognize the same T7 and SP6 transcription promoters as standard T7 and SP6 RNA Polymerases.

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.

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.

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.

Figure 3 (click to enlarge). 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.