Hi all,
I have been trying to synthesize luciferase self-amplifying RNA (saRNA) by in vitro transcription (IVT), and this vector has approximately 11kb. The main problem is that I am getting a product of approximately 8kb. (p.s. I am using the NEB HiScribe kit)
I have linearised my plasmid with MluI and the bands on the gel seem to be fine (see the picture attached). Upon completing my RNA IVT, I usually get a good RNA yield that is around 800-1000 ng/ul.
The main problem seems to be the premature termination of the polymerase, hence the shorter RNA product. Does anyone know if there could be other causes for this? Or how to prevent premature termination?
Many thanks,
Beatriz
I second Guillermo's suggestion. Try running your saRNA on a denaturing gel with an ssRNA ladder if you want to get the most accurate sizing. Agarose gels will also work, but you'll still need to use the ssRN ladder to size your IVT product.
Hi Guillermo Posadas-Herrera and John Hardy Lockhart
Many thanks for your replies. I have actually run the denaturing gel with the RNA ladder, should have included the picture here. Please find it attached now.
As you can see, my band is around 8kb, and not 11kb as expected. I've used RNA millenium marker, thus top band is 9kb.
Your bands seem quite clean, so it doesn't seem like the polymerase is simply falling off randomly.
Are you sure that the RNA product should be 11 kb? I expect it to be a few kb shorter than the plasmid since you won't include the parts necessary for plasmid replication and bacterial selection during IVT.
John Hardy Lockhart yes, I've done these IVTs before and I was getting bands at 11kb. When I started using these new kits, I started noticing this.
I get very efficient reactions, good yield, and good pellet, but the RNA is not of the right size. I am not sure if it is something with the new kit or something in the process (linearisation, IVT, purification?)
I've used the NEB HiScribe® T7 High Yield RNA Synthesis Kit in my previous work to make self-replicating mRNAs (also around 11 kb) without any issues. The yields for these longer mRNAs were of course lower, but I don't recall ever having issues with premature termination.
Are you using a cap analog or performing capping enzymatically after synthesis?
John Hardy Lockhart I am capping enzymatically after synthesis with the ScriptCap™ Cap 1 Capping System. Do you think it could be related to it?
Beatriz Dias Barbieri
I was actually going to suggest the enzymatic approach if you were using an analog, as the cap analogs can interfere with IVT, particularly in longer synthesis reactions.
The only other things I can think of that might be causing issues is some secondary structure that is stopping the T7 polymerases or there is a T7 terminator (or similar) sequence somewhere in your gene.
You can try incubating the IVT reaction at 30 ˚C to increase the processivity of the T7 polymerase, but your yields will decrease.
Assuming that your plasmid template is exactly as you expect it to be, the final thing to try is to sequence the IVT product. You can use oligo-dT primers on your polyA-tailed IVT mRNA and design sequencing probes upstream of the suspected termination site.
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