EZ-QC™ mRNA Cap 1 Efficiency Assay Kit
- Quantitate the percentage of mRNA with Cap 1 vs Cap 0 5’-cap
structures using standard polyacrylamide gel electrophoresis (PAGE).
- Easy, cost-effective benchtop method for QC as an alternative to mass spec analysis.
Product Description
The EZ-QC™ mRNA Cap 1 Efficiency Assay Kit quantitates the percentage
of Cap 1 vs Cap 0 5’-cap structures in an mRNA production product. The
5’ RNA cap consists of an N7-methylated guanine nucleoside joined to the
RNA via a 5’ to 5’ triphosphate linkage. This reaction is catalyzed by
mRNA Capping Enzyme (e.g., ScriptCap™ Capping Enzyme)
and produces a Cap 0 cap structure. Cap 0 caps can be converted into
Cap 1 caps by the additional treatment of an mRNA 2’-O-Methyltransferase
(e.g., ScriptCap™ 2’-O-Methyltransferase)
which catalyzes the 2’-O-methylation of the ribose sugar of the first
base of the RNA transcript. Most Cap 1 mRNAs are expressed at higher
levels in cells, relative to their Cap 0 counterparts since the Cap 1
helps to identify the mRNA as "self" RNA in cells and thus not elicit an
innate immune response against the mRNA.1,2
The EZ-QC™ mRNA Cap 1 Efficiency Assay Kit reaction labels the mRNA 5'-caps with Cyanine5 Hydrazide (Cy5®).
The mRNA 5'-uncapped ends are not labeled. The labeled mRNA is then
hydrolyzed with an RNase mixture releasing Cy5-labelled Cap 1 and Cap 0
cap cores which are resolved using polyacrylamide gel electrophoresis
and analyzed using a fluorescent gel imager to measure the relative
amounts of Cap 1 to Cap 0 present in the sample. The assay simplifies
analysis and replaces tedious and expensive capping efficiency
determination methods such as HPLC and mass spectrometry.
For determination of total percent capped RNA content, CELLSCRIPT™ also offers theEZ-QC™ mRNA Capping Efficiency Assay Kit(distinguishes between total capped [Cap 0 Cap 1] and uncapped RNAs) and theEZ-QC™ mRNA Poly(A) Tail Length Assay Kits for complete mRNA characterization.
References:
- Daffis, S. et al., (2010) Nature 468, 452.
- Devarkar, S.C. et al., (2016) PNAS 113, 596.
Product Performance
The EZ-QC™ mRNA Cap 1 Efficiency Assay Kit evaluates the efficiency
of the methylation event catalyzed by 2’-O-Methyltransferase, which
converts Cap 0 structures to Cap 1 structures. Labeled cap cores can be
resolved on a polyacrylamide gel and quantified using fluorescent gel
imaging software and the included 80/20 Cap 1 Control Mix (Figure 1).
Figure 1. Cap
0, Cap 1, and a mixture of Cap 0 and Cap 1 mRNA assessed using the
EZ-QC™ mRNA Cap 1 Efficiency Assay Kit. Gel imaging software was used to
quantitate the percentage of Cap 0 and Cap 1 mRNA in each lane.
| Lane |
Percent Cap 1 |
Percent Cap 0 |
|
1 |
78.1 |
21.9 |
|
2 |
79.2 |
20.8 |
|
3 |
0 |
100 |
|
4 |
100 |
0 |
|
5 |
78.8 |
21.2 |
Materials Supplied
Important Upon receipt of the kit, remove the
reagents and store at the indicated temperatures. Store at -20°C in a
freezer without a defrost cycle. Do not store at –70°C.
EZ-QC™ mRNA Cap 1 Efficiency Assay Kit (10 reactions)
Sufficient for 10 experimental and 10 control reactions. |
|
Kit Component |
Reagent Volume |
Storage Temperature |
|
1 M Sodium Acetate |
20 μl |
Ambient |
|
4 mM Sodium Sulfite |
20 μl |
Ambient |
|
10 mM Sodium Periodate |
40 μl |
4°C |
80/20 Cap 1 Control Mix
Contains a mix of 80% Cap 1 and 20% Cap 0 mRNAs |
60 μl |
–20°C |
|
5 mM Cy5 Hydrazide (in DMF) |
20 μl |
–20°C |
EZ-QC™ RNase T1
in 50% glycerol and 50 mM Tris-HCl, pH 7.4 |
20 μl |
–20°C |
EZ-QC™ RNase I
in 50% glycerol, 50 mM Tris-HCl, pH 7.5, 100 mM NaCl and 0.1 mM EDTA |
24 μl |
–20°C |
Cap 1 Stop/Loading Dye
95% formamide, 0.4 mM EDTA and 40 μg/ml Basic Fuchsin |
350 μl |
–20°C |
|
RNase-Free Water |
875 μl |
–20°C |
Materials Required, but not Supplied
- Purified capped mRNA.
- Materials for polyacrylamide gel electrophoresis.
- RNAClean XP beads (Beckman Coulter) for mRNA clean-up.
- 70% ethanol
- 0.2-ml polypropylene thermocycler-compatible tubes with caps or 96-well 0.1-ml PCR plate with microseal plastic films. Note: Cy5 hydrazide is provided in DMF, which is incompatible with polystyrene-based consumable materials.
- Materials and equipment for polyacrylamide gel visualization, imaging and quantification capable of Cy5 emission detection.
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