Applications

In vitro Transcription: INCOGNITO™ IVT and mRNA Production Systems

The new INCOGNITO™ kits

Evade cellular immune response to your RNA with the new INCOGNITO™ kits from Cellscript.

The new INCOGNITO™ kits * are designed for in vitro synthesis of RNA containing modified nucleosides such as pseudouridine and 5-methylcytidine. They assist in reducing immune response and enhancing protein translation. A range of these kits are available to end users depending on their specific research desires and downstream applications.

Cellscript’s INCOGNITO™ contain everything needed to make N1meΨ- or Ψ-containing Cap1 mRNA, including in vitro transcription reagents plus ScriptCap™ Capping Enzyme, ScriptCap™ 2’-O-Methyltransferase, and A-Plus™ Poly(A) Polymerase.

The INCOGNITO™ T7-FlashScribe™ N1meΨ-RNA Transcription Kit is specially formulated to enable users to obtain the maximum possible yields of N1-methyl-pseudouridine-containing RNA (N1meΨ-RNA) from an in vitro transcription (IVT) reaction. Although yield varies with the DNA template and other factors, a standard 60 minute, 20 μl reaction will yield up to 160 μg of RNA from 1 μg of the control template. These yields are made possible by the high-performance properties of the T7-FlashScribe™ enzyme. The standard reaction can be scaled up to produce milligram amounts of RNA containing the canonical nucleotides ATP, CTP, GTP and the modified nucleotide N1-methyl-pseudouridine-5'-triphosphate (N1meΨTP).

The INCOGNITO™ T7-FlashScribe™ Ψ-RNA Transcription Kit is specially formulated to maximize yields of pseudouridine-containing RNA (Ψ-RNA) from an in vitro transcription (IVT) reaction. Although yield varies with the DNA template and other factors, a standard 60 minute, 20 μl reaction will yield up to 160 μg of RNA from 1 μg of DNA template. These impressive yields are due to the high-performance properties of the T7-FlashScribe™ enzyme. The standard reaction can be scaled up to generate milligram amounts of RNA containing the canonical nucleotides ATP, CTP, GTP and the modified nucleotide pseudouridine-5'-triphosphate (ΨTP).

INCOGNITO™ T7 mScript™ mRNA Production Systems contain everything needed to make N1meΨ- or Ψ-containing Cap1 mRNA, including in vitro transcription reagents plus ScriptCap™ Capping Enzyme, ScriptCap™ 2’-O-Methyltransferase, and A-Plus™ Poly(A) Polymerase.

The INCOGNITO™ SP6 Ψ-RNA Transcription Kit -optimized for high-yield synthesis of pseudouridine-containing RNA (Ψ-RNA) from an in vitro transcription (IVT) reaction.

The INCOGNITO™ T7 5mC- & Ψ-RNA Transcription Kit - optimized for high-yield synthesis of 5-methyl-cytidine- pseudouridine-containing RNA (5mCΨ-RNA) from an in vitro transcription (IVT) reaction.

Advantages

Optimized for high-yield synthesis of pseudouridine-containing IVT RNA (Ψ-RNA)
Produce up to 100 µg of Ψ-RNA from 1 µg of template ( depending on kit used).
Easily scale up reactions to produce milligram amounts of Ψ-RNA.
Lower immunogenicity in mammalian cells through incorporation of Ψ in synthesized mRNA.
Modified Ψ-mRNAs translate into higher protein levels
The standard reactions can be scaled up to produce milligram amounts of RNA canonical nucleotides ATP and GTP and modified pseudouridine-5'-triphosphate (ΨTP) and 5-methyl-cytidine-5'-triphosphate (5mCTP).

*Transcription Kits are for in vitro transcription of N1meΨ- or Ψ-containing RNA. - All kits above come with a label license to use them for internal research use under the University of Pennsylvania’s modified mRNA technologies patents.

INCOGNITO™ T7-FlashScribe™ Ψ-RNA Transcription Kit

CellScript

Catalogue No.	Description	Pack Size	Price	Qty	Change to: €
IFY240525	INCOGNITO™ T7-FlashScribe™ Ψ-RNA Transcription Kit	25 rxns	POA	Quantity	Add to Order

Description

Optimized for maximum yields of pseudouridine-containing IVT RNA
(Ψ-RNA).
Produce up to 160 µg of Ψ-RNA from 1 µg of template in just one hour.
Easily scale up reactions to produce milligram amounts of Ψ-RNA.
Lower immunogenicity in mammalian cells through incorporation of Ψ in synthesized mRNA.

Product is for research use only (RUO)

Product Description:

The INCOGNITO™ T7-FlashScribe™ Ψ-RNA Transcription Kit is specially formulated to maximize yields of pseudouridine-containing RNA (Ψ-RNA) from an in vitro transcription (IVT) reaction. Although yield varies with the DNA template and other factors, a standard 60 minute, 20 μl reaction will yield up to 160 μg of RNA from 1 μg of DNA template. These impressive yields are due to the high-performance properties of the T7-FlashScribe™ enzyme. The standard reaction can be scaled up to generate milligram amounts of RNA containing the canonical nucleotides ATP, CTP, GTP and the modified nucleotide pseudouridine-5'-triphosphate (ΨTP).

Modified Ψ-mRNAs translate into higher protein levels and lower innate immune responses in human and other mammalian cells that express various RNA sensors compared to unmodified mRNAs.

INCOGNITO™ T7 IVT Ψ-RNA can be further processed into low immunogenicity mRNA (5'-end capped and 3'-end poly[A] tailed) through the use of CELLSCRIPT™'s ScriptCap™ Cap 1 Capping System (contains both ScriptCap™ Capping Enzyme and 2'-O-Methyltransferase) and A-Plus™ Poly(A) Polymerase Tailing Kit (available separately).

For maximum reduction of immunogenicity, combine INCOGNITO™ Ψ-mRNA with the Min-Immune™ Gold dsRNA Removal Kit to produce virtually dsRNA-free, ultra-low immunogenicity mRNA that is suitable for downstream applications, such as cell and gene therapy research and mRNA vaccine development.

Product Performance:

The standard 60 minute, 20 μl reaction was optimized for transcription using 1 µg of linearized DNA template. However, incubation times can be adjusted if desired. Table 1 summarizes transcription results with 1 µg of the control template DNA in a standard reaction with incubation times from 20-120 minutes. Results may vary depending on the template used.

Table 1. IVT RNA yields from INCOGNITO™ T7-FlashScribe™ Ψ-RNA Transcription Kit reactions incubated between 20-120 minutes. The standard protocol uses an incubation time of 60 minutes.

Incubation Time (minutes)	20	30	60	90	120
RNA Yield (µg)	55-92	102-128	150-190	150-205	152-208

Uses 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 Licensing tab for more information.

Materials Supplied

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

INCOGNITO™ T7-FlashScribe™ Ψ-RNA Transcription Kit Contents (25 reactions)
Kit Component	Reagent Volume
T7-FlashScribe™ Enzyme Solution	50 μl
10X T7-FlashScribe™ Transcription Buffer II	50 μl
100 mM GTP	45 μl
100 mM ATP	45 μl
100 mM ΨTP	45 μl
100 mM CTP	45 μl
100 mM Dithiothreitol (DTT)	50 μl
RNase-Free DNase I, 1 U/μl	25 μl
ScriptGuard™ RNase Inhibitor, 40 U/μl	15 μl
T7 Control Template DNA, 0.5 μg/μl	10 μl
RNase-Free Water	1.4 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
RNase-free TE Buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA)

Terms and Conditions: The Product listed is currently offered by CELLSCRIPT™ for research use only under the defined Terms and Conditions.

Troubleshooting guide:

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 4 hours.
	Increase the template concentration.
	Increase the reaction temperature to 42^oC.
	Check for stable secondary structure (T_m >37^oC) in the DNA template which can cause T7 RNAP pausing or dissociation from the template.
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.
White precipitate in reaction buffer	Do not store the kit at –70^oC.

FAQs: For answers to Frequently Asked Questions about our products, visit the FAQ library.