Dear Samuel,

Thank you for your question. The inclusion of modified bases in a transcription can alter several parameters. If you are including ARCA in the reaction and reducing the GTP (e.g. 4:1 ARCA:GTP) in order to give good capping, this will reduce your expected transcription yields by about 3-4 fold. In our hands, full substitution of U and C with pseudo-U and 5-methyl-C do no significantly reduce transcription yields. For an ARCA cappedmRNA  fully substituted with pseudo-U and 5-methyl-C, we typically see crude yields of 1.5-2 mg/ml of transcription. We optimize transcription reactions by varying the MgCl₂ concentration. 4-6 mM over total NTPs usually works well.

With regards to the 260/280 ratio, the extinction coefficients and lambda maxes of pseudo-U and 5-methyl C differ from U and C. The lambda max for pseudo-U is 265 vs 262 and 5-Methyl-C is 279 vs 271. Thus, pseudo-U and 5-methyl-C substitution would be predicted to give you a higher 260/280 ratio than normal NTPs. If you are observing a lower 260/280 ratio, this might mean that you are actually doing a better job of removing protein in this prep. I would not be concerned by this.

With regard to mobility, we do sometimes see that some modifications change the mobility of the RNA on gels. Base modification can change the secondary structure of the mRNA and thus the mobility on non-denaturing gels. We recommend glyoxal treating the mRNA with NorthernMax®-Gly Loading Dye and then running it with NorthernMax® 10X Running Buffer (do not use TAE or TBE).

I hope this information is helpful. If you continue to have problems, you could considering order a custom mRNA.

Best regards,
Anton McCaffrey, PhD
Principal Scientist