EZ-QC™ mRNA Capping Efficiency Assay Kit

Quantitate mRNA 5’ capping efficiency of mRNA with any known 5’ UTR sequence using standard polyacrylamide gel electrophoresis (PAGE).
Generate same-day percent capped RNA quality control results in your lab.
Experience cost-savings through use of common laboratory equipment, rather than specialized instrumentation.

Product is for research use only (RUO)

Product Description:

The EZ-QC™ mRNA Capping Efficiency Assay Kit provides quantitative capping efficiency (percent capped RNA content) analysis of synthesized mRNA. Since only capped RNAs are expressed in cells, it is essential to have the highest possible percentage of capped RNAs present in a sample. The EZ-QC™ mRNA Capping Efficiency Assay Kit simplifies analysis and replaces tedious and expensive capping efficiency determination methods such as HPLC and mass spectrometry, instead allowing for determination based upon standard polyacrylamide gel electrophoresis (PAGE) methodology.

The EZ-QC™ mRNA Capping Efficiency Assay utilizes a chimeric RNA-DNA-RNA Targeting Oligonucleotide (user provided) which hybridizes to the 5'-end of an RNA mixture. This hybridization complex serves as a substrate for RNase H cleavage releasing the capped and uncapped 5'-end fragments which can subsequently be resolved via PAGE and quantified by gel band analysis. The method allows for straightforward, fluorescence-based calculation of the percent capped RNA content of the sample. The specificity of the Targeting Oligonucleotide, designed to facilitate cutting the mRNA at a precise location, provides greater confidence in determining the percentage of capped/uncapped content of the assayed RNA sample.

Note: The EZ-QC™ mRNA Capping Efficiency Assay does not differentiate between Cap 0 and Cap 1 capped RNAs, only between capped (Cap 0 + Cap 1) and uncapped RNAs.

Alternatively, the EZ-QC™ XBG mRNA Capping Efficiency Assay Kit is also designed for determination of percentage capped RNA content and provides an XBG 5' UTR Targeting Oligonucleotide. For determination of Cap 0 vs Cap 1 content, CELLSCRIPT™ also offers the EZ-QC™ mRNA Cap 1 Efficiency Assay Kit 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.

Product Performance:

EZ-QC™ mRNA Capping Efficiency Assay Targeting Oligonucleotide design considerations

The EZ-QC™ mRNA Capping Efficiency Targeting Oligonucleotide should be designed to release an mRNA 5'-end fragment of 25-40 nucleotides in length (Figure 1). This released fragment, +/– the cap nucleotide, is to be resolved via PAGE analysis. RNase H cleavage will occur in the mRNA strand across from the 5'-most DNA base in the Targeting Oligonucleotide.

Figure 1. Graphic representation of mRNA and Targeting Oligonucleotide.

Resolution of capped and uncapped 5’-end fragments

Mixtures of capped and uncapped mRNA were assayed using the EZ-QC™ mRNA Capping Efficiency Assay Kit and resolved on a polyacrylamide gel (Figure 2A). The Targeting Oligonucleotide facilitates cutting the mRNA at a single, repeatable location allowing for greater confidence in determining the percentage of capped and uncapped content of the assayed samples. The percent capped and uncapped mRNA in a mixture are calculated with a simple readout from any gel imaging system (Figure 2B).

Figure 2A. Capped and uncapped 5’-end fragments are efficiently resolved by PAGE allowing for capping efficiency analysis with standard lab equipment.

Figure 2B. Densitometric data from lanes 4, 5, and 6 from the above PAGE gel using a Syngene^® G:Box Gel Documentation System. The percent capped and uncapped mRNA in each mixture are calculated using a simple readout.

Materials Supplied:

Important Store at -20°C in a freezer without a defrost cycle. Do not store at –70°C.

EZ-QC™ mRNA Capping Efficiency Assay Kit (10 reactions) Sufficient for 10 experimental and 10 control reactions.
Kit Component	Reagent Volume
EZ-QC™ RNase H in 50% glycerol, 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT), 0.1 mM EDTA and 0.1% Triton® X-100.	20 μl
10X EZ-QC™ RNase H Reaction Buffer 0.2 M Tris-acetate, pH 7.9, 0.5 M potassium acetate, 0.1 M magnesium acetate and 0.01 M DTT.	20 μl
ScriptGuard™ RNase Inhibitor, 40 U/μl in 50% glycerol, 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM DTT, 0.1 mM EDTA and 0.1% Triton X-100.	10 μl
Stop/Loading Buffer 95% formamide, 10 mM EDTA, pH 7.5, 0.01% Bromophenol Blue and 0.01% Xylene Cyanol	200 μl
RNase-Free Water	500 μl

Materials Required, but not Supplied

Purified capped mRNA/RNA
EZ-QC™ mRNA Capping Efficiency Targeting Oligonucleotide
Materials for polyacrylamide gel electrophoresis
Materials for polyacrylamide gel visualization, imaging and quantification
Optional: molecular weight marker (e.g., ssDNA 10/60 Ladder [IDT])

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

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Troubleshooting Guide:

Symptom	Solution
More than two bands present in the capped/uncapped region of the gel	The Targeting Oligo hybridized in multiple locations. Redesign the Targeting Oligo.
A DNA-only Targeting Oligo was used. Use a chimeric RNA-DNA-RNA Targeting Oligo.
Targeting Oligo migrates within the capped/uncapped excised fragment range. Redesign Targeting Oligo to either be of a different size than the capped/uncapped excised fragments or to hybridize in a different position such that the capped/uncapped excised fragments are of a different size than the Targeting Oligo.
Faint capped/uncapped bands	mRNA 5'-end secondary structure is preventing efficient hybridization of the Targeting Oligo. Preanneal the mRNA and Targeting Oligo, via mixing, heating and cooling, before adding the rest of the reaction components. Redesign the Targeting Oligo to hybridize to a different region of the mRNA. Sometimes shifting the RNase H cut site over by as little as one base can be helpful.
One or more of the four DNA bases in the chimeric Targeting Oligo are hybridizing to a pseudouridine or N1-methyl-pseudouridine base on the mRNA. Redesign Targeting Oligo to hybridize to a different region where the DNA bases of the Targeting Oligo will hybridize only to non-pseudouridine or non-N1-methyl-pseudouridine bases.
A molar excess of Targeting Oligo was not used in the reaction. Unhybridized Targeting Oligo should be visible on the gel. Recheck experimental stoichiometry. Add more Targeting Oligo to the reaction.
Load more completed reaction sample per well.
Increase the reaction incubation time to 60 minutes.
Near full lane of many bands	RNase contamination. Be sure to use ScriptGuard™ RNase Inhibitor in the reaction. Decontaminate tubes, pipet tips and surfaces. Always use gloved hands.
Working stock of Targeting Oligo is degrading. Use or make a different working concentration aliquot of the Targeting Oligo.
Fuzzy bands	Problems with the PAGE system (e.g., gel not totally polymerized, gel ran too hot, urea wasn't blown out of the wells just prior to sample loading, buffer issue). Rerun the gel.
Capped and uncapped bands unresolved	A <20% polyacrylamide gel was used. Rerun the gel using 20% polyacrylamide. Commercially-available pre-poured 15% gels most often do not resolve the bands adequately.

Symptom

Solution

More than two bands present in the capped/uncapped region of the gel

The Targeting Oligo hybridized in multiple locations.

Redesign the Targeting Oligo.

A DNA-only Targeting Oligo was used.

Use a chimeric RNA-DNA-RNA Targeting Oligo.

Targeting Oligo migrates within the capped/uncapped excised fragment range.

Redesign Targeting Oligo to either be of a different size than the capped/uncapped excised fragments or to hybridize in a different position such that the capped/uncapped excised fragments are of a different size than the Targeting Oligo.

Faint capped/uncapped bands

mRNA 5'-end secondary structure is preventing efficient hybridization of the Targeting Oligo.

Preanneal the mRNA and Targeting Oligo, via mixing, heating and cooling, before adding the rest of the reaction components.
Redesign the Targeting Oligo to hybridize to a different region of the mRNA. Sometimes shifting the RNase H cut site over by as little as one base can be helpful.

One or more of the four DNA bases in the chimeric Targeting Oligo are hybridizing to a pseudouridine or N1-methyl-pseudouridine base on the mRNA.

Redesign Targeting Oligo to hybridize to a different region where the DNA bases of the Targeting Oligo will hybridize only to non-pseudouridine or non-N1-methyl-pseudouridine bases.

A molar excess of Targeting Oligo was not used in the reaction. Unhybridized Targeting Oligo should be visible on the gel.

Recheck experimental stoichiometry.
Add more Targeting Oligo to the reaction.

Load more completed reaction sample per well.

Increase the reaction incubation time to 60 minutes.

Near full lane of many bands

RNase contamination.

Be sure to use ScriptGuard™ RNase Inhibitor in the reaction.
Decontaminate tubes, pipet tips and surfaces. Always use gloved hands.

Working stock of Targeting Oligo is degrading.

Use or make a different working concentration aliquot of the Targeting Oligo.

Fuzzy bands

Problems with the PAGE system (e.g., gel not totally polymerized, gel ran too hot, urea wasn't blown out of the wells just prior to sample loading, buffer issue).