EZ-QC™ mRNA Poly(A) Tail Length Assay Kit
- Quantitative mRNA poly(A) tail length using standard polyacrylamide gel electrophoresis (PAGE).
- Easy, cost-effective benchtop method for QC as an alternative to mass spec analysis.
- Assess tail lengths above the limits of HPLC and LC-MS based methods.
Product Description
The EZ-QC™ mRNA Poly(A) Tail Length Assay Kit provides quantitative
mRNA poly(A) tail length analysis of synthesized mRNA. 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. 3'-poly(A) tails can be added
co-transcriptionally (utilizing a template-encoded tail) or
post-transcriptionally (via enzymatic addition) to in vitro
transcribed (IVT) RNA as a step in the mRNA synthesis process. The
EZ-QC™ mRNA Poly(A) Tail Length Assay Kit simplifies analysis and
replaces tedious poly(A) tail length determination methods requiring
reverse transcription, PCR or RNase H digests. The kit uses RNase A to
digest non-adenine residues in the mRNA molecule, leaving the poly(A)
tail intact to allow for length determination based upon standard
polyacrylamide gel electrophoresis (PAGE) methodology.
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.
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