- Precise mRNA poly(A) tail length quantitation using standard polyacrylamide gel electrophoresis (PAGE).
- Assess tail lengths from 20 to > 500 bases, above the limits of
LC-MS analysis, using the provided proprietary Poly(A) 20-mer Ladder.
- Time- and cost-saving, same day mRNA poly(A) tail QC at your own benchtop.
Product is for research use only (RUO)
Product Description
The EZ-QC™ mRNA Poly(A) Tail Length Assay Kit provides precise
fluorescence-based quantitative mRNA poly(A) tail length analysis of
synthesized mRNA. Tail lengths even greater than 500 bases, above the
limits achievable by LC-MS analysis (i.e. 9-156 bases), can be assessed
using standard polyacrylamide gel electrophoresis (PAGE). 3'-end
polyadenylation, the addition of a poly(A) tail to mRNA, is a critical
modification that influences mRNA stability and expression levels in
eukaryotic cells, with longer tails generally providing increased
stability and translation efficiency. The EZ-QC™ mRNA Poly(A) Tail
Length Assay Kit simplifies analysis and replaces tedious poly(A) tail
length determination methods requiring RT-PCR or specialized
instrumentation. The assay uses RNase A to selectively digest
non-adenine residues in the mRNA sample, leaving only the poly(A) tail
intact. Length determination is completed using a PAGE methodology
enhanced with the proprietary Poly(A) 20-mer Ladder, allowing for high
resolution quantitation of poly(A) tails even above the limits of LC-MS
analysis.
For determination of percent capped RNA content, CELLSCRIPT™ also offers the EZ-QC™ mRNA Capping Efficiency Assay Kit and the EZ-QC™ mRNA Cap 1 Efficiency Assay Kit for complete mRNA characterization.
Product Performance
Sample input
Using the standard protocol and an average image exposure time of
~0.5 seconds, as little as 1 pmol of input poly(A)-tailed RNA yields a
signal visible by eye (Figure 1). Utilizing gel imaging and analysis
software (Syngene® G:Box & GeneTools), as little as 0.1
pmol of input poly(A)-tailed RNA yields a detectable and measurable
signal. Increased exposure times or increased sample volume loading can
be used to further minimize the amount of input poly(A)-tailed RNA
required. The protocol is robust. Input poly(A)-tailed RNA amounts can
be easily modified depending on sample availability.
Figure 1. Input
titration of EZ-QC™ mRNA Poly(A) Tail Length Assay Kit. As little as
0.1 pmol mRNA input is detectable and measurable using gel analysis
software.
Quantitative Detection
A single run can resolve a wide variety of different tail lengths and
sample tail types. Figure 2A shows the Poly(A) 20-mer Ladder (lanes 1
and 6), an encoded 120 nt tail (lane 2), a 15-minute enzymatic 136 nt
tail (lane 3), a 30-minute enzymatic 193 nt tail (lane 4) and a
60-minute enzymatic 261 nt tail (lane 5) within a single gel run. The
gel shows both the level of tailing as well as the distributive
properties of enzymatic tailing. The tail length is accompanied by a
correlation in the overall peak uniformity and signal intensity to help
determine optimal enzymatic conditions (Figure 2B).
Figure 2A:
Polyacrylamide gel showing poly(A) tail lengths ranging from 120 nt to
261 nt from the EZ-QC™ mRNA Poly(A) Tail Length Assay Kit
Figure 2B: Distribution of signal from analysis of the polyacrylamide gel. Average tail lengths are calculated.
Downloads
Manual
SDS
Components list
Materials Supplied
Important Store at -20°C in a freezer without a defrost cycle. Do not store at –70°C.
EZ-QC™ mRNA Poly(A) Tail Length Assay Kit (10 reactions)
Sufficient for 10 experimental and 10 control reactions (optional) |
|
Kit Component |
Reagent Volume |
EZ-QC™ RNase A
in 50% glycerol, 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA and 0.1% Triton® X-100. |
20 μl |
10X EZ-QC™ RNase A Reaction Buffer
0.6 M Tris-HCl, pH 8.0, 1 M NaCl, 0.1 M EDTA. |
20 μl |
Stop/Loading Buffer
95% formamide, 10 mM EDTA, pH 7.5, 0.01% Bromophenol Blue and 0.01% Xylene Cyanol. |
200 μl |
Poly(A) Fluorescence Enhancer, 100X
Protect the tube from exposure to light. |
3 x 1.7 ml |
Poly(A) 20-mer Ladder
Ready-to-Load: supplied in Stop/Loading Buffer |
100 μl |
|
RNase-Free Water |
500 μl |
Materials Required, but not Supplied
- Purified poly(A)-tailed mRNA/RNA
- Materials for polyacrylamide gel electrophoresis.
- Materials for polyacrylamide gel visualization, imaging and quantification.
If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200