Messenger RNA can be used for protein expression into the cells. The delivery of mRNA is the delivery of a new function that can activate a biological process. In the particular case of the CAS9 mRNA, it is simple way to make gene editing. Messenger RNAs are at the center of attention to fight cancers and for personalized medicine. However, production of capped mRNA can be a challenge. Let’s see how to face up to it here with some tips.
High yield in vitro transcription
The best yields are obtained with T7 promoter. Indeed, it is good to avoid SP6 and T3 when possible.
The highest performances are around 180µg of mRNA from 1µg of DNA template. The T7-FlashScribe can provide 172µg in only 30 minutes as shown below.
A polyA tail in 3′ of the mRNA is required. The expression vector can include it. I would suggest 120A as for our catalogue mRNA for which we have a good feedback. However, short version such as 80 polyA is also functional
Close to 100% capping in one reaction
A new nucleotide, called CleanCap AG reagent, can ensure +96% capping during the in vitro transcription without additional reactions. The CleanCap AG reagent is used similarly to the famous ARCA, but provides more capping efficiency as illustrated below.
Furthermore, the capping structure is a CAP1 with Clean Cap AG vs a basic CAP0 using ARCA. CAP1 is known to boost protein production into the cells.
How do I proceed?
Just combine the CleanCap Ag reagent, 040N-7113-10 or 040N-7413-10, and the T7-FlashScribe kit (for 50 reactions). The detailed reaction is below.
In vitro transcription reaction
Warning: The T7 promoter must be with an AGG initiator. So the T7 promoter must be 5’TAATACGACTCACTATAAGGNNNNNNNNNNNN… 3’. Our lab can offer to modify your promoter as service.
We can also prepare your mRNA to bring you complete peace of mind if you prefer.
Get in touch through the form below with your questions, we’ll be pleased to get back to you with answers and solutions!
2 Responses
Hi,
I got a problem regarding low yield of mRNA.
I prepared fresh IVT buffer and 2.5ug DNA template for 100ul Rxn and used N1-Methylpseudo-UTP, ATP, CTP, and GTP (5ul for each) with CleanCap AG (4ul) for 3 hours incubation in the thermal cycler.
And then purify mRNA by RNeasy Mini Kit (QIAGEN).
However, the yield is only 30ug per 100ul Rxn which is quietly lower than the expected yield on protocol.
Dear Chih-Jen,
Thank you for your question on our blog.
I recommend we discuss this further by email.
Here are my contact details: isabelle.nobiron@tebu-bio.com.
Looking forward to hearing from you.
Kind regards,
Isabelle