Product Description:
The Min-Immune™ Gold dsRNA Removal Kit provides a novel enzymatic solution for the removal of double-stranded RNA (dsRNA) contamination present in RNA samples produced by in vitro transcription (IVT). dsRNA can be reduced to <0.005% (LLOQ) of the sample, lower than what can be achieved using engineered mutant T7 RNA polymerases. Each 25-reaction kit will treat 1,500 μg of IVT RNA or mRNA and is suitable for most samples regardless of length, end modifications or NTP content.
The removal of dsRNA from mRNA preparations has been shown to be essential for reducing the innate immunogenic response to the mRNA in cells. Alternative dsRNA removal methods, such as reverse-phase HPLC, hydroxyapatite chromatography and cellulose chromatography, are associated with high capital costs as well as reduced final product yields. The Min-Immune™ Gold dsRNA Removal Kit provides an easy to use, scalable method for removing the dsRNA content of IVT RNA or mRNA preps without a reduction of the single-stranded RNA yield. Use of Min-Immune™ Gold in combination with incorporation of modified nucleosides into in vitro transcribed mRNA can serve as a highly efficient workflow for maximum reduction of immunogenicity for downstream applications, such as cell and gene therapy research and mRNA vaccine development.
Product Performance:
dsRNA reduction to <0.005% (LLOQ)
A Min-Immune™ Gold dsRNA Removal Kit treated 1.4 kb pseudouridine-containing RNA sample was compared to an untreated sample (Figure 1, right panel) and dsRNA Standards (Figure 1, left panel). Triplicates of each sample and standard were immobilized on a charged nitrocellulose membrane, then immunoblotted with a primary dsRNA-specific antibody and a secondary antibody conjugated to horseradish peroxidase (HRP). Signal was detected using enhanced chemiluminescence on a Syngene® G:Box. A regression equation was generated using the dsRNA standard curve on the left and is used to calculate the percent dsRNA present in both the treated and untreated samples seen on the right side of the blot. Results show a reduction of dsRNA content in the treated sample replicates to levels below the lower limit of quantification (0.005%) of this assay after treatment with the Min-Immune™ Gold dsRNA Removal Kit.
Figure 1. Immunoblot of Min-Immune™ Gold dsRNA Removal Kit treated RNA. dsRNA is reduced below the lower limit of quantification after treatment.
Treatment of RNA with inherent stable dsRNA regions
The Min-Immune™ Gold dsRNA Removal Kit is designed to remove contaminating dsRNA generated during transcription from IVT RNA and mRNA preparations. However, some RNAs naturally contain dsRNA regions or structures within them. The lengths of these dsRNA regions dictate their sensitivity to Min-Immune™ Gold RNase III cleavage as well as to recognition by intracellular dsRNA specific pattern recognition receptors. In general, Min-Immune™ Gold RNase III will recognize hairpin structures starting at 17 bp for uridine-containing RNA and 22 bp for pseudouridine-containing RNA. Digestion efficiency will increase with longer hairpin lengths (Figure 2). Because of this, it is recommended to characterize Min-Immune™ Gold treatment of all new RNA samples with a 1X or smaller reaction prior to treatment of larger amounts of the same RNA.
Figure 2. Seven constructs were designed to contain inherent dsRNA regions of 12-26 bp long hairpins in the secondary structure of the resultant RNAs. Following transcription with canonical nucleosides (GAUC, left panel) or including the modified nucleoside pseudouridine (GAΨC, right panel), the RNA samples were treated with the Min-Immune™ Gold dsRNA Removal Kit and visualized on a formaldehyde-denaturing 1% agarose gel. Duplexes of 15-bp dsRNA or shorter were not recognized by Min-Immune™ Gold RNase III within GAUC-RNA, while duplexes of 19-bp dsRNA and shorter were not recognized within GAΨC-RNA.
Treatment of RNA with transient dsRNA regions
Some RNA samples can form transient dsRNA regions (molecular breathing) during the reaction which are substrates for Min-Immune™ Gold RNase III cleavage. These will appear as faint, less than full length background bands when visualized on a gel (Figure 3). This clipping does not affect the bulk functionality of the treated RNA. If minor clipping occurs, subsequent samples of the RNA can be treated more gently by reducing the incubation time or amount of Min-Immune™ Gold RNase III used.
Figure 3. A pseudouridine-containing mRNA sample (Ψ-mRNA) was treated using the Min-Immune™ Gold dsRNA Removal Kit and visualized on a formaldehyde-denaturing 1% agarose gel. Faint background bands from clipping of transient dsRNA sites can be seen when the image is purposefully overexposed. This clipping does not affect the bulk functionality of the treated RNA.
Materials Supplied:
Important Store at -20°C in a freezer without a defrost cycle. Do not store at –70°C.
Min-Immune™ Gold dsRNA Removal Kit Contents (25 reactions) |
Kit Component |
Reagent Volume |
Min-Immune™ Gold RNase III (20X)
in 50% glycerol, 50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1 mM dithiothreitol (DTT), 0.1 mM EDTA and 0.1% Triton® X-100. |
150 μl |
Min-Immune™ Gold 10X RNase III Treatment Buffer |
300 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. |
75 μl |
5 M Ammonium Acetate |
2 x 1.6 ml |
RNase-Free Water |
2 x 1.7 ml |
Materials Required, but not Supplied
- mRNA or IVT RNA for treatment
- 70% ethanol
Optional Materials
dsRNA-specific detection system including:
- dsRNA-specific antibody (e.g., J2 antibody [Absolute Biotech-Exalpha])
- Dot/slot blotting system for use with the antibody
- An image analyzer for blot visualization and/or quantification
- dsRNA standards
Terms & Conditions: The Product listed is currently offered by CELLSCRIPT™ for research use only under the defined Terms and Conditions.
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 |
Solution |
Faint, minor, less than full length background bands are seen on the gel post-treatment |
RNA samples were not fully denatured.
- Be sure to be running a denaturing agarose gel.
- Rerun the samples with an increased heat denaturing step prior to gel loading.
|
RNA sample formed transient dsRNA regions (molecular breathing) during the reaction which were substrates for Min-Immune™ Gold RNase III cleavage.
- If very minor, no further treatment nor treatment changes are needed.
- If minor, treat subsequent samples of this RNA more gently. Reduce incubation to 30 minutes or reduce the amount of Min-Immune™ Gold RNase III used by 50%.
|
Strong, major, less than full length background bands are seen on the gel post-treatment |
RNA sample contained an inherent stable dsRNA region of sufficient length to be a substrate for Min-Immune™ Gold RNase III cleavage.
- The Min-Immune™ Gold dsRNA Removal Kit is not appropriate for treating this particular RNA sequence.
If the dsRNA structure is crucial to RNA functionality, use an alternative method for dsRNA removal. If the dsRNA structure is not crucial to RNA functionality, mute the dsRNA structure to remove the duplex formation by altering the RNA sequence and retreat the new RNA. |
dsRNA assessment indicates remaining dsRNA content |
RNA contains a region of dsRNA.
- dsRNA region may be inherent to the RNA sequence and recognizable by the dsRNA-specific antibody but may not be recognizable by the Min-Immune™ Gold RNase III or intracellular dsRNA-specific pattern recognition receptors.
- If very minor, no further treatment nor treatment changes are needed.
- If minor, the RNA sample may be retreated with Min-Immune™ Gold RNase III. If the dsRNA assessment still shows dsRNA content after retreatment, then the dsRNA region is most likely inherent to the RNA sequence. Try the RNA in the downstream application. If the dsRNA assessment now shows virtually no dsRNA, the next time that specific RNA is to be made, more strongly treat the RNA by extending the incubation time to 2 or 3 hours or increasing the amount of Min-Immune™ Gold RNase III used in the reaction.
|
Abnormally high amounts of dsRNA were present in the RNA sample.
- Retreat the RNA sample.
- The next time that specific RNA is to be made, more strongly treat the RNA by extending the incubation time to 2 or 3 hours or increasing the amount of Min-Immune™ Gold RNase III used in the reaction.
- Consider using an altered transcription protocol that would produce less dsRNA during the IVT reaction.
|
Min-Immune™ Gold dsRNA Removal reaction was inefficient or failed.
|
LIMITED LABEL LICENSE
CELLSCRIPT™’s Min-Immune™ Gold dsRNA Removal Kit (“Product“) effectively removes double-stranded RNA (dsRNA) contaminants that are generated as by-products of making mRNA using a process comprising In vitro transcription (IVT) of a DNA template encoding said mRNA. This Product and the compositions and methods and uses of any thereof are covered by United States and International Patents and Patent Applications including and derived from World Patent Organization PCT Patent Application Number WO 2013/102203A1 and U.S. Patent Number US12059479B2, and further patents that issue from divisional and continuation patent applications thereof.
By purchasing Product from CELLSCRIPT™ or an authorized distributor of CELLSCRIPT™ (herein, “Purchaser”), Purchaser receives a limited non-exclusive, non-transferable, non-sublicensable right (“Limited License”) to use the purchased Product solely for its own internal laboratory research use (the “Licensed Use”) and Purchaser agrees to cite the name of the Product and CELLSCRIPT™ as its source in any verbal or written public disclosure about said Licensed Use. The Licensed Use expressly excludes any Commercial Use comprising [A] any diagnostic, prophylactic, clinical, therapeutic or other use in humans, [B] any veterinary, livestock, agricultural use in animals, and/or [C] manufacture, distribution, importation, exportation, or sale of any other products and/or services made using Product for which Purchaser receives compensation of any kind, it being understood that [A]–[C] are separately and/or collectively defined as a “Commercial Use” herein. No other license rights are granted, expressly or implied, to make, have made, import, export, use, reverse engineer, transfer, offer for sale, or sell Product or any other CELLSCRIPT™ product or service. By purchase of Products, Purchaser agrees not to make, have made, import, export, use, reverse engineer, transfer, offer for sale, or sell Products, components of Products, or derivatives of Products, or to provide services, information, or data obtained by the use thereof in exchange for money or other consideration. CELLSCRIPT™ provides no warranties (statutory or implied) concerning non-infringement of intellectual property rights of any other parties, and all such warranties are expressly disclaimed. This Limited License terminates automatically if Purchaser breaches any of the terms herein.
The license remains in effect if the product is being used in accordance with the license terms. The license is terminated immediately upon user’s breach of any of the license terms, such as using the product for commercial purposes without proper authorization.
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