The EZ-QC™ XBG mRNA Capping Efficiency Assay Kit provides
quantitative capping efficiency (percent capped RNA content) analysis of
synthesized mRNA containing a Xenopus beta-globin (XBG) 5' UTR
(untranslated region). 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™ XBG 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™ XBG mRNA Capping Efficiency Assay utilizes a chimeric
RNA-DNA-RNA Targeting Oligonucleotide (kit provided) which hybridizes to
the 5'-end region of an RNA mixture when the mRNA construct contains a
Xenopus beta-globin 5' UTR. 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™ XBG 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™ mRNA Capping Efficiency Assay Kit is
also designed for determination of percentage capped RNA content but
requires the end user to provide their own chimeric RNA-DNA-RNA
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.
Resolution of capped and uncapped 5’-end fragments
Mixtures of capped and uncapped mRNA were assayed using the EZ-QC™
XBG mRNA Capping Efficiency Assay Kit and resolved on a polyacrylamide
gel (Figure 1A). 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
1B).
Figure 1A. Capped and uncapped 5’-end fragments
are efficiently resolved by PAGE allowing for capping efficiency
analysis with standard lab equipment.
Figure 1B. 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™ XBG mRNA Capping Efficiency Assay Kit Contents (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. |
23 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. |
23 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. |
12 μl |
XBG 5' UTR Targeting Oligo, 5 μM (5 pmoles/μl)
in RNase-Free Water. |
28 μl |
XBG 5' UTR Control Mix, 2.5 μM (2.5 pmoles/μl)
an 80% capped / 20% uncapped RNA Mix in RNase-Free Water. |
6 μl |
Stop/Loading Buffer
95% formamide, 10 mM EDTA, pH 7.5, 0.01% Bromophenol Blue and 0.01% Xylene Cyanol. |
230 μl |
|
RNase-Free Water |
575 μl |
Materials Required, but not Supplied
- Purified capped mRNA/RNA
- 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.
Troubleshooting Guide:
|
Symptom |
Solution |
|
More than two bands present in the capped/uncapped region of the gel |
The Targeting Oligo hybridized in multiple locations.
- Run the capped and uncapped experimental samples alongside a kit
control reaction to identify the experimental bands of interest, then
quantitate only the bands of interest.
Note: the released 5'-end fragments from the experimental
mRNAs may not comigrate with those of the control reaction depending on
the sequence of the experimental mRNAs. |
|
Targeting Oligo migrates within the capped/uncapped excised fragment range.
- Run the capped and uncapped experimental samples alongside a lane
containing the XBG 5' UTR Targeting Oligo-only to identify the
experimental bands of interest, then quantitate only the bands of
interest.
|
|
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.
|
|
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.
|
|
XBG 5' UTR Targeting Oligo is degrading.
- Run a sample of the XBG 5' UTR Targeting Oligo-only on a gel to assay for integrity.
|
|
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).
|
|
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.
|
If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200