EZ-QC™ mRNA Poly(A) Tail Length Assay Kit

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™ XBG mRNA Capping Efficiency Assay Kit (for mRNA with an XBG 5’ UTR), EZ-QC™ mRNA Capping Efficiency Assay Kit (for mRNA with any known 5’ UTR sequence) and EZ-QC™ mRNA Cap 1 Efficiency Assay Kit for complete mRNA characterization. For more information about EZ-QC™ mRNA QC technologies, visit our mRNA QC analysis hub.

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.

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
EZ-QC™ 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

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	Example	Solution
Light sample: Poly(A) tail band and Poly(A) 20-mer Ladder are both faint		Poly(A) Fluorescence Enhancer was not used during the gel staining process. • Restain the gel including Poly(A) Fluorescence Enhancer.
		Insufficient gel staining time. • Return gel to stain solution for a longer period of time. NOTE: The 100-bp DNA marker band will stain more readily than the poly(A) bands. Use the intensity of this band to determine proper remedy.
		Excessive dilution of sample with Stop/Loading Buffer. • Use a 1:1 ratio of reaction sample and Stop/Loading Buffer.
		Insufficient sample volume loaded on the gel. • Load ≥2 μl of stopped reaction sample per lane
Light sample: Light or absent poly(A) tail bands but Poly(A) 20-mer Ladder is not faint		Insufficient amount of mRNA used in the reaction. • Use 5 pmol of mRNA in the reaction. Signals generated from ≤ 2 pmol of mRNA produce a faint and harder to identify band. However, instrumentation may still be sensitive enough to detect at this level. • Load more reaction sample per lane.
Near full lane of many bands		RNase A digestion failure. • Repeat RNase A treatment.
Secondary (smaller) bands seen following digestion		The higher molecular length band is the RNase A-resistant poly(A) tails of the mRNA (blue dot). The lower molecular length band is the RNase A-resistant poly(A) tails of very short secondary (byproduct) transcripts generated during the transcription step of mRNA synthesis (red dots). These small transcripts may be sense (i.e., abortive transcripts) or antisense (generated by RNA-dependent, promoter-independent mechanisms during transcription, and become poly(A) tailed during the tailing step of mRNA synthesis). Running mRNA that has not been treated with RNase A, Lane 3, (green dot) on the gel alongside treated samples (Lane 2) will confirm the presence of tailed short fragments as they are only slightly longer than the RNase A treated version of these fragments. Note: Short fragments are not an indication of failure of the EZ-QC™ mRNA Poly(A) Tail Length Assay, but are instead, a byproduct of in vitro transcription.