EZ-QC™ mRNA Capping Efficiency Assay Kit

A:No, the EZ-QC™ mRNA Capping Efficiency Assay Kit can only determine if a sample is capped (Cap 0 or Cap 1) or uncapped. To quantitate the percent Cap 1 and percent Cap 0 content of your mRNA production product, CELLSCRIPT™ offers the EZ-QC™ mRNA Cap 1 Efficiency Assay Kit.

A:Yes, the assay has been tested successfully up to 50 pmol of input sample mRNA.

A:Yes, it will.

A: No, the targeting oligo must be an RNA:DNA:RNA chimeric oligo with 4 DNA bases where the desired cut should be. If a targeting oligo only contains DNA bases this will result in several cleavage sites and the resulting gel image will have multiple bands complicating interpretation.

Figure 1: Chimeric vs DNA targeting oligo. UC=Uncapped RNA and C=Capped RNA

A: Standard desalting purification is all that is required. Higher levels of purification are not necessary.

A: It is recommended to design the targeting oligo with a T_m range between 50-68°C.

A: The DNA bases of the EZ-QC™ mRNA Capping Efficiency Targeting Oligonucleotide should not be designed such that they will hybridize with a pseudouridine/N1-methyl-pseudouridine in the mRNA. However, it is fine if the RNA bases of the targeting oligo do.

A: The EZ-QC™ RNase H cleavage efficiency will be reduced, but you still might be able to generate adequate quality data for capping efficiency determination.

A: The best resolution was found with 20% acrylamide. Even with 17% the band separation is difficult to visualize.

A: Yes, if the pre-cast is 20% acrylamide.

A: Products are good for up to a month if stored at -20°C.

A: This would suggest the sample is 100% capped or 100% uncapped.

A: At most there should be 3 bands; capped mRNA, uncapped mRNA, and the targeting oligo at the bottom. If there are greater than this number of bands check to make certain the targeting oligo is a RNA:DNA:RNA hybrid. If the targeting oligo is DNA only this will lead to multiple bands and confound interpretation.