T7 mScript™ Complete Standard mRNA Production System

Complete solution to produce uridine-containing, fully 5’ capped and
3’ poly(A) tailed mRNA (U-mRNA) with post-transcriptional enzymatic dsRNA removal to <0.005% (LLOQ).
Minimize immunogenicity in mammalian cells by synthesizing transcripts followed by highly efficient post-transcription removal of dsRNA.
Produce 1.5 mg of U-mRNA with virtually 100% transcript 5’ capping.
Create variable 3’ poly(A) tail lengths, from 50 to >300 A’s.

Product is for research use only (RUO)

Use and Label Licenses for Specific Products: Purchaser receives a limited non-exclusive, non-transferable right to use the Products purchased from CELLSCRIPT™ solely for its own internal laboratory research use. See the Label License tab for more information.

Product Description:

The T7 mScript™ Complete Standard mRNA Production System provides all enzymes and reagents for making low immunogenicity uridine-containing (GAUC),
5'-capped, 3'-polyadenylated mRNA with ultra-low double-stranded RNA (dsRNA) content. The kit includes reagents for five workflow modules, (1) high-yield in vitro transcription of linear double-stranded DNA templates using the T7 mScript™ Enzyme Solution and the canonical nucleotides ATP, CTP, GTP and UTP, (2) post-transcriptional enzymatic capping of the RNA using the ScriptCap™ Cap 1 Capping System (contains both ScriptCap™ Capping Enzyme and 2'-O-Methyltransferase) for making mRNA with a Cap 1 cap structure, (3) A-Plus™ Poly(A) Polymerase for adding a 3'-poly(A) tail, (4) post-transcriptional enzymatic removal of dsRNA content using the Min-Immune™ Gold dsRNA Removal system and (5) 5 M NH₄OAc as a convenient RNA/mRNA purification method.

Post-transfection, 5’ capped and 3’ poly(A) tailed mRNA has increased stability and translation efficiency in most eukaryotic cell lines. The mScript™ System improves upon co-transcriptional capping and tailing methods by ensuring virtually 100% transcript capping and enabling users to define their desired tail lengths. The A-Plus™ Poly(A) Polymerase protocol provides a wide range of poly(A) tail lengths from 150–300+ bases. The included Min-Immune™ Gold dsRNA Removal module can reduce dsRNA to <0.005% (LLOQ) of the sample, lower than what can be achieved using engineered mutant T7 RNA polymerases. Low immunogenicity T7 mScript™ Complete U-mRNA is suitable for use in downstream applications such as cell and gene therapy research and mRNA vaccine development.

For maximum reduction of immunogenicity, the INCOGNITO™ T7 mScript™ Complete N1meΨ-mRNA Production System and INCOGNITO™ T7 mScript™ Complete Ψ-mRNA Production System produce virtually dsRNA-free, ultra-low immunogenicity mRNA containing the modified nucleotides N1-methyl-pseudouridine and pseudouridine, respectively.

Materials Supplied:

Important Store at -20°C in a freezer without a defrost cycle. Do not store at –70°C.

T7 mScript™ Complete Standard mRNA Production System Kit Contents 25 Reactions. (Module 1 of 5)
Kit Module	Kit Component	Reagent Volume
In Vitro Transcription	T7 mScript™ Enzyme Solution	50 µl
	10X T7 mScript™ Transcription Buffer II	50 µl
	100 mM Dithiothreitol (DTT)	50 µl
	NTP Solution 25 mM each GTP, ATP, UTP, CTP	180 µl
	RNase-Free DNase I, 1 U/μl	25 µl

T7 mScript™ Complete Standard mRNA Production System Kit Contents 25 Reactions. (Module 2 of 5)
Kit Module	Kit Component	Reagent Volume
Post-Transcriptional Capping	ScriptCap™ Capping Enzyme, 10 U/μl	100 µl
	ScriptCap™ 2'-O-Methyltransferase, 100 U/μl	100 µl
	10X ScriptCap™ Capping Buffer 0.5 M Tris-HCl (pH 8.0), 60 mM KCl and 12.5 mM MgCl₂	250 µl
	20 mM S-adenosyl-methionine (SAM)	125 µl
	20 mM GTP	125 µl

T7 mScript™ Complete Standard mRNA Production System Kit Contents 25 Reactions. (Module 3 of 5)
Kit Module	Kit Component	Reagent Volume
Poly(A) Tailing	A-Plus™ Poly(A) Polymerase, 4 U/µl	130 µl
	10X A-Plus™ Poly(A) Tailing Buffer 0.5 M Tris-HCl (pH 8.0), 2.5 M NaCl and 100 mM MgCl₂	300 µl
	20 mM ATP	150 µl

T7 mScript™ Complete Standard mRNA Production System Kit Contents 25 Reactions. (Module 4 of 5)
Kit Module	Kit Component	Reagent Volume
dsRNA Removal	Min-Immune™ Gold RNase III (20X)	150 µl
dsRNA Removal	Min-Immune™ Gold 10X RNase III Treatment Buffer	300 µl

T7 mScript™ Complete Standard mRNA Production System Kit Contents 25 Reactions. (Module 5 of 5)
Kit Module	Kit Component	Reagent Volume
Common Usage	ScriptGuard™ RNase Inhibitor, 40 U/µl	165 µl
	RNase-Free Water	12 ml
	5 M Ammonium Acetate	12 ml

T7 Control Template DNA: Is a linearized 4.1 kb plasmid that contains a T7 promoter followed by a phage lambda dsDNA insert that encodes a 1,375 base runoff transcript. The Control Template DNA is provided at a concentration of 0.5 µg/µl in T₁₀E₁ Buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA).

Materials Required, but not Supplied

A DNA template for transcription of your RNA of interest
Materials or kits for purification of the RNA product if not using the kit provided NH₄OAc
RNase-free TE Buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) for final mRNA resuspension-only, if not using the kit provided RNase-Free Water
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
TE saturated phenol/chloroform, 0.5-1 M EDTA

→ Manual

→ SDS

→ Product Flyer

→ CELLSCRIPT™ mRNA Production Systems Comparison Table

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:

Synthesis of IVT RNA
Symptom	Solution
Low yields or less than full-length transcripts	Cleanup the templates to remove any RNase or other contaminants.
	Verify that ScriptGuard™ RNase Inhibitor was added to the reaction.
	Extend the incubation time. Do not extend the reaction time beyond 3 hours.
	Increase the template concentration.
	Increase the reaction temperature to 42^oC.
Assembled reaction formed an insoluble precipitate	Repeat assembly of the reaction at >22^oC.
White precipitate in reaction buffer	Incubate the reaction buffer at 37^oC for 5 minutes then mix thoroughly to dissolve the precipitate.
	Do not store the kit at –70^oC.

Synthesis of Capped RNA
Symptom	Solution
Low capping efficiency	Cleanup the templates to remove any RNase or other contaminants.
	Verify that ScriptGuard™ RNase Inhibitor was added to the reaction.
	SAM slowly degrades at room temperature and above. Keep SAM solutions on ice at all times.
	Increase the capping reaction incubation time. For example, up to 3 hours at 37^oC.
	Some RNAs form stable structures (e.g., homodimers, hairpins) at the 5' end, limiting access by Capping Enzyme or 2'-O-Methyltransferase. Analyze the sequence and increase the RNA denaturation temperature to above the T_m (e.g., to 65^oC for 20 min, 75^oC for 10 min, 85^oC for 5 min). If the 5' end is highly structured, it might be necessary to modify the 5' end sequence using molecular biology techniques. Often this can be accomplished by making a single point mutation within the first 5 bases of the DNA template for the RNA transcript (non-coding region). Contact CELLSCRIPT™ Technical Support for suggestions and recommendations.
White precipitate in reaction buffer	Incubate the reaction buffer at 37^oC for 5 minutes then mix thoroughly to dissolve the precipitate.
	Do not store the kit at –70^oC.

Synthesis of Poly(A)-Tailed RNA
Symptom	Solution
Poly(A) tails are longer than expected	Decrease the time of incubation of the reaction.
	Decrease the amount of A-Plus™ Poly(A) Polymerase used in the reaction.
Poly(A) tails are shorter than expected	Increase the time of incubation of the reaction.
	Increase the amount of A-Plus™ Poly(A) Polymerase used in the reaction.
No poly(A) tails are observed	Enzyme is inactive. Store only at –20^oC. Keep on ice when not in the freezer.
	ATP is hydrolyzed. Do not expose to elevated temperatures.

dsRNA Removal
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 system 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. Retreat the RNA sample. If the problem persists Contact CELLSCRIPT™ Technical Support.

LIMITED LABEL LICENSE

CELLSCRIPT™’s T7 mScript™ Complete Standard mRNA Production System (“Product“) is for making standard (U)-mRNA having reduced innate immunogenicity in mammalian cells. This Product and the compositions and methods and uses thereof are covered by WIPO PCT Patent Application Numbers WO2007024708A2 and WO2011071931A2 and U.S. Patent Numbers US8278036B2 and US9750824B2, and U.S. and international divisional, continuation, or other patents and patent applications derived therefrom that are owned by the University of Pennsylvania and licensed to CELLSCRIPT™.

The Min-Immune™ Gold dsRNA Removal Module of this Product effectively removes double-stranded RNA 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 Module and the compositions and methods and uses thereof are covered by WIPO PCT Patent Application Number WO2013102203A2 and U.S. Patent Number US12059479B2 and U.S. and international divisional, continuation, or other patents and patent applications derived therefrom that are owned by CELLSCRIPT™.

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, [C] making or using U-mRNA encoding transcription factors or cell reprograming factors to generate human induced pluripotent stem cells (iPSCs), hematopoietic stem or progenitor cells, neurons, immune cells or any other differentiated cells, and/or [D] 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]–[D] 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.

If your organization is interested in using T7 mScript™ Complete Standard mRNA Production System or any other CELLSCRIPT™ products for a Commercial Use, whether in catalog or custom sizes, please contact customerservice@cellscript.com with a description of your interest and needs.