RiboMAX Large Scale RNA Production Systems (SP6 and T7) From Promega

I used this kit to produce RNA for injecting into Xenopus embryos. In order to transcribe RNA, your DNA template should be linearized prior to in vitro transcription. After linearization, DNA should be cleaned either by phenol extraction followed by ethanol precipitation, or using any clean-up columns. I used Wizard® DNA Clean-Up System (Promega Cat.# A7280). Dilute the resulting DNA in double-distilled sterile water and test its quality by agarose gel and and its concentration by spectrophotometer.

The transcription reaction for SP6 or T7 RNA Polymerase is set up at room temperature. I needed to produce capped RNA for injection into the Xenopus embryos. For this, the final GTP concentration is reduced to 0.6 mM and m7G(5´)ppp(5´)G is added to a final concentration of 3 mM. Usually, I mixed equal amounts of ATP, CTP, UTP nucleotides together and GTP to get 3 mM. So, in my stock, I had 25 mM each of ATP, CTP, UTP and 3 mM GTP. After preparing the stock, I aliquoted and stored it at -20°C. Then, I mixed the reaction components: SP6 or T7 Transcription 5X Buffer, rNTPs (25mM ATP, CTP, GTP, UTP), linear DNA template (5-10 µg total), Enzyme Mix (SP6 or T7) plus Nuclease-Free Water up to chosen total volume of the reaction. Note that reaction volume may vary from 20 μl up to 100 μl. Usually, the reaction volume depends of the template DNA concentration. In my system, reaction worked well in reaction volumes up to 50 μl.

I found out that heating DNA to 70°C for 5 minutes prior to setting up the reaction really helped to produce more and better quality RNA. These reactions can be scaled up or down to suit your template requirements. I usually used different amounts RNA: from less than 5 μg to more then 10 μg, and succeeded in every one of them. Incorporating a cap analog may reduce the yield of RNA to 20-50% of a standard reaction. The ratio of cap analog:GTP is 5:1 in the protocol, but can be varied from 10:1 to 1:1 to balance the percentage of capped products with the efficiency of the transcription reaction.

Gently mix the reaction by pipetting it up and down a few times and incubate at 37°C for 2-4 hours. I didn't find that prolonged incubation increased the yield of RNA, but addition of 0.5 μl of the chosen enzyme after 1 hour of incubation really helped to get more RNA. For my purposes, I had to remove DNA. I used RQ1 RNase-Free DNase to a concentration of 1 unit per 1 µg of template DNA immediately after the end of the transcription reaction. I incubated the reactions 15 minutes at 37°C, then cleaned the RNA by phenol:chlorophorm extraction followed by ethanol precipitation. I reconstituted the RNA in double-distilled sterile water and tested its quality by agarose gel and its concentration by spectrophotometer. Usually, I obtained RNA transcripts of good quality and concentration. This is a reliable kit; it produces good quality RNA and I highly recommend it.