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).
Generate same-day mRNA Cap 1 efficiency quality control results in your lab.
Experience cost-savings through use of common laboratory equipment, rather than specialized instrumentation.
Note: The EZ-QC™ mRNA Cap 1 Efficiency Assay labeling chemistry is not compatible with cap structures that contain a 3'-O-methyl group on the capping G nucleotide such as Anti Reverse Cap Analog (ARCA) or some versions of GpppAG cap analogs.

Product is for research use only (RUO)

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 sample. 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" and lowers the innate immune response against the mRNA in cells. The EZ-QC™ mRNA Cap 1 Efficiency Assay Kit quantifies the conversion efficiency of Cap 0 caps into Cap 1 caps by an mRNA 2’-O-Methyltransferase (e.g., ScriptCap™ 2’-O-Methyltransferase).

The EZ-QC™ mRNA Cap 1 Efficiency Assay Kit reaction labels mRNA 5'-caps with Cyanine5 (Cy5) Hydrazide, which leaves any mRNA 5'-uncapped ends unlabeled. The labeled capped 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 (PAGE). A standard fluorescent gel imager measures the relative amounts of Cap 1 to Cap 0 present in the sample for accurate calculation of Cap 1 conversion efficiency. 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 the EZ-QC™ XBG mRNA Capping Efficiency Assay Kit (for mRNA containing an XBG 5’ UTR) and EZ-QC™ mRNA Capping Efficiency Assay Kit (for mRNA containing any known 5’ UTR sequence) as well as the EZ-QC™ mRNA Poly(A) Tail Length Assay Kit for complete mRNA characterization. For more information about the EZ-QC™ mRNA quality control technologies, visit our mRNA QC analysis hub.

Note: The EZ-QC™ mRNA Cap 1 Efficiency Assay labeling chemistry is not compatible with cap structures that contain a 3'-O-methyl group on the capping G nucleotide such as Anti Reverse Cap Analog (ARCA) or some versions of GpppAG cap analogs.

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).

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

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.

Materials Supplied:

Important Upon receipt of the kit, remove the reagents and store at the indicated temperatures.

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

Terms & Conditions: The Product listed is currently offered by CELLSCRIPT™ for research use only under the defined Terms and Conditions.

→ Manual

→ SDS

→ Product Flyer

FAQs: For answers to Frequently Asked Questions about our products, visit the FAQ library.

Still have questions? Our mRNA experts are here to help.

Troubleshooting Guide:

Symptom	Solution
Experimental bands are not visible but the 80/20 Cap 1 Control Mix bands are	Insufficient amount of input mRNA was used. If performing bead-based clean up, ensure that ≥12 pmol of mRNA are used for the assay. Requantify the mRNA stock to ensure that 12-24 pmol of mRNA are used for the assay. Loss of purified mRNA pellet. If performing precipitation-based clean up, pellets from 12-24 pmol of mRNA may be difficult to see by eye and may be lost during cleanup if not handled carefully. Repeat the reaction taking special care at this step. The overall capping efficiency of the mRNA prep was low. The kit detects levels of Cap 1 relative to Cap 0, not relative to the total mRNA content. If the overall percent capped mRNA content is low, fewer cap core molecule are labeled resulting in a weak signal. To determine total percent capping, use the EZ-QC™ mRNA Capping Efficiency Assay Kit or the EZ-QC™ XBG mRNA Capping Efficiency Assay Kit (for mRNA containing a Xenopus β-globin [XBG] 5' UTR).
Experimental and the 80/20 Cap 1 Control Mix bands are not visible	Reaction failure. Repeat the reaction using fresh input mRNA. Sodium periodate was not reactive. Repeat the reaction with a fresh dilution of the sodium periodate stock.
Background bands are visible higher in the gel than the area of the Cap 1/Cap 0 bands of interest	The Cy5 labeling reaction will label all nucleic acid molecules containing vicinal hydroxyl groups. This includes the 3’-most base of the mRNA and any carried over nucleotides (NTP, NDP, NMP) that were part of the transcription or capping or tailing reactions, which were not removed during the labeled mRNA clean up step. The presence of these bands does not change the accuracy of the assay and they should be disregarded during the quantitation analysis step.
Background bands are visible in the area of the Cap 1/Cap 0 bands of interest	A background band appearing slightly above the Cap 1 or Cap 0 bands of interest can be an indication of a low efficiency N7-methylation reaction on the capping G nucleotide which would have been catalyzed by the Capping Enzyme. Use the cumulative signal intensity of both bands as the calculated intensity of the appropriate cap core band for quantitation calculations. If desired, the mRNA can be recapped to complete the N7-methylation. Verify that the S-adenosyl-methionine used for this step is still intact.
The percentage Cap 1/Cap 0 calculated by the imaging software doesn't seem to match with what is observed by visual inspection of the gel image	The dynamic range of the imaging detector has been exceeded during the imaging process. The gel was over-exposed such that the most intense bands are out of the quantification range. Under-exposure of the gel may also impact quantitation. Ensure that gel imaging exposure times are such that no pixels are saturated and that bands are adequately exposed.
	Depending on the amount of input mRNA used for the assay, and depending on how efficient the Cap 1 methylation reaction was during RNA capping, there can be little to no Cap 0 present in the mRNA prep. For mRNA preps with ≥95%, but <100% Cap 1 content, detection of the Cap 0 band may depend on the sensitivity of the gel imaging equipment.
Only background bands are visible, cap core bands are not visible, band migration is toward the bottom of the gel	Gel composition and/or running buffer composition may be incorrect.