I am setting up in vitro reaction by T7 polymerase on linearized plasmids purified by Promega column after digestion. Some people recommend using phenol;chloroform to clean DNA before transcription. My transcripts are long, between 1 to 2 kb. Though the RNA band on the gel looks intact, the yield is very low. How to increase the yield of RNA. My reaction with final concentration is as follows:
40 mM Tris–HCl pH 7.9, 6 mM MgCl2,10 mM DTT, 2 mM spermidine, 0.5 mM each rATP/rCTP/rUTP and 0.1 mM rGTP, 0.5 mM RNA cap-analog. 40 U RNasin, 50U T7 RNA polymerase, about 500ng of DNA in 20ul reaction, put for 4 hours at 37-40C. Please suggest how to increase the yield, thanks.
Hi,
you can try different things and should optimize the reaction for all of your transcripts.
You can try over night up to 16 hours or find the optimal temperature. Do you use a kit or mix these things on your own? the amount of DTT can be reduced as it possibly hinders the polymerase. 1mM should be enough to loosen up secondary structures. There is no standard protocol which functions for all transcripts as it depends on the composition and lenght of the transcript (GC-cont.)
I have a detailed protocol somewhere but I need to have a look...
Sachin, do you add more rGTP, up to total 0.5mM, after starting the reaction with cap-analog for 15 min? I don't see this passage in your description, but it is crucial for your RNA elongation after forcing the 5' capping for a short while
Both from my experience and the composition of kits aimed at increasing short RNA IVT yield, I would say that increasing the enzyme-to-template ratio in combination with the two-step (low rGTP --> high rGTP) suggested by Fabrizio should significantly boost your yields. Good luck!
Hi Fabrizio and Anna,
Thanks! What I understood is to increase T7 polymerase enzyme amount in the reaction, isn't it? Cap is a limiting factor in transcribing long capped mRNAs. And most protocols ask for using Cap:rGTP in the ratio of at least 4:1. I was keeping rGTP to 0.1mM throughout the reaction. My concern is if I increase up to 0.5mM rGTP after 15 min of start, then most mRNAs produced after that will remain uncapped. NEB manual says that at the ratio of 1:1 Cap:rGTP (which it will become after keeping rGTP to 0.5mM) about 50% of RNA will be capped and at the ratio of 5:1, more than 80% will be capped. These transcripts are viral mRNAs which I have to rub-inoculate on plants to get virus infection, so more the capped, better it is. However, RNA yield is another important factor for successful reaction. I can increase rGTP to 0.5 and then increase the cap-analog to 1mM, that way the ratio will be 2:1 and 67% RNA should be capped in that case. What do you suggest? Thanks.
Another alternative to the GTP chase that Fabrizio suggested is to forgo the cap analog and add the cap enzymatically after the IVT step. NEB has a Vaccinia capping system, https://www.neb.com/products/m2080-vaccinia-capping-system that works really well in my experience. Leaving the cap analogs out of the IVT allows higher NTP levels and greatly boosts your overall yield. 1-2kb length should not be limiting for IVT; we routinely perform up to 4kb transcripts and do not observe truncation.
Really it depends of the structure of your mRNA. I guess that you want to study the translation of the capped mRNA. If so, the structure of the 5'UTR has to be looked at. Maybe you could precise the structure of your inserted gene to be transcribed. Did you try to translate your RNA without any capping. Some in vitro transcribed RNAs work very well without any capping.
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