The kit utilizes CleaScrip^TM T7 RNA Polymerase (Low dsRNA) and high-affinity LZcap AG Cap1 analog to produce mRNAs with significantly reduced double-stranded RNA (dsRNA) content and enhanced expression. Single-stranded RNAs are synthesized using linear double-stranded DNA containing a T7 promoter sequence as the template. Modified nucleotides, such as biotin or dye-labeled nucleotides, can be incorporated during transcription to generate labeled RNA. The kit supports the synthesis of both long and short transcripts, yielding 100–200 μg of RNA from 1 μg of DNA template. Substituting UTP with N1-Me-Pseudo UTP reduces RNA immunogenicity. The resulting RNA is suitable for downstream applications, including RNA structure and function studies, RNase protection, probe hybridization, RNAi, microinjection, and in vitro translation.

Components

Note: The T7 RNA Polymerase Mix includes CleaScrip^TM T7 RNA polymerase (Low dsRNA, Cat: 10628), Murine RNase inhibitor (Cat: 10621) and Pyrophosphatase Inorganic (Cat: 10620); The Cap1 Analog is LZcap AG (Cat: 10684). The catalog number of recommended UTP and Pseudo UTP is 10654 and 10656, respectively.   

Figure

Figure 1. The Structure of LZCap AG.

Table 1. Co-transcription analysis of the input of LZcap AG. IVT materials include CleaScrip T7 RNA Polymerase, Template: Cas9 (4K); T7 Reaction Buffer 2; Tris NTP (including N1-Me-Pseudo UTP Tris Solution).  IVT time: 3 hrs.

Table 2. dsRNA content analysis of regular T7 Kit and Low dsRNA T7 Kit.

Storage

This product should be stored at -25 ~-15℃ for one year.

Experimental methods

1. DNA template preparation

Linearized plasmids with double-stranded T7 promoters or PCR amplification products can be used as T7 High Yield RNA Synthesis Kit for Co-transcription in vitro transcription templates, which can be dissolved in TE buffer or RNase free H₂O. T7 promoter sequence: TAATACGACTCACTATAA*GG (Note: A* is the first base of RNA transcription).

A. Plasmid template

Insert the target DNA to the plasmid vector containing the T7 promoter, and then treated with restriction enzymes, purified after completely linearized.

Notes:

 The circular plasmids have no effective termination, RNA products of different lengths will be transcribed. In order to obtain a specific length RNA, the plasmid must be completely linearized.

 The restriction enzyme selected for plasmid linearization needs to be on the right side of the promoter region, downstream of the inserted DNA fragment, and has no recognition site in the inserted DNA fragment. The restriction enzyme should be capable of forming 5' sticky ends or smooth ends.

 In order to avoid the influence of protein and salt ions on the system, the plasmid is recommended to be purified when used as a template for in vitro transcription after linearization.

B. PCR product template

The PCR product with T7 promoter can be used as an in vitro transcription template. First, add the T7 promoter sequence (TAATACGA CTCACTATAAGG) to the 5' end of the upstream primer sense strand; next, the T7 promoter DNA template is amplified under the action of high fidelity enzyme; then transcription is performed. PCR products can be used directly as templates without purification, but higher RNA output will be obtained after purification.

Notes:

 The specificity and concentration of the PCR product must be confirmed by electrophoresis when used as a template. Put 2-5 μL of PCR product into the 20 μL reaction system.

 In order to obtain more high-quality RNA, the PCR product should be recovered by gel and used as a template for in vitro transcription.

2. In vitro RNA co-transcription

A. Thawing reagents

Centrifuge the T7 RNA Polymerase Mix briefly and place on ice. Thaw 10×Transcription Buffer, ribonucleotides (ATP, CTP, GTP, N¹-Me-Pseudo UTP) and CAP1 GAG, then centrifuge to the bottom of the tube, place 10× Transcription Buffer at room temperature, and place others on ice.

B. Assembly transcription reaction at room temperature

Prepare the reaction system according to the following system:

Notes:

 The reaction is configured at room temperature. Since 10×Transcription Buffer contains spermidine, the concentration of spermidine too high will cause DNA template precipitation at low temperature.

 Short transcript (<100 nt),2 μg template can be used, transcription time increased to 4-8 hs.

 For long transcripts (>1000 nt), recommended to use linearized plasmid templates for transcription.

Perform the reaction in a PCR machine with the hot lid open to prevent the reaction solution from evaporating for a long time.

 The reaction product may have a white precipitate. This is free pyrophosphate and magnesium ions produce the magnesium pyrophosphate in the reaction, won’t affect the subsequent experiments. You can add some EDTA to clear it. If the addition of EDTA affects subsequent experiments, the supernatant can also be recovered by centrifugation.

 The reagents and containers must without RNase contamination.

C. Incubate at 37°C for 2-3 hours

Mix the above reaction solution, briefly centrifuge to the bottom of the tube, and incubate at 37°C for 3 hs. If the transcript length is less than 100 nt, increase the reaction time to 4-8 hs.

D. DNase I treatment (optional)

After the reaction is complete, add 1 μL of DNase I  to each tube and incubate at 37°C for 15 mins to remove the template DNA.

3. Product purification

The transcribed RNA can be precipitated by lithium chloride solution in this kit, then remove proteins and free nucleotides. The purified RNA can be subjected to downstream experiments or stored at -80°C after electrophoresis detection. The lithium chloride precipitation method demands the RNA length must be greater than 300 nt, and the concentration must not be less than 100 ng/μL.

①Add 30 μL RNase free H₂O and 30 μL 7.5 M lithium chloride to 20 μL reaction mixture.

②After mixing uniformly, place it at -20°C for at least 30 mins, centrifuge at 4°C for 15 mins at maximum speed, and collect the precipitate.

③Add 500 μL of ice-cold 70% ethanol to wash the RNA pellet.

④Dissolve the RNA pellet with 20 μL RNase free H₂O. The purified RNA solution is stored at -80°C.

4. RNA quantification

A. Ultraviolet absorption

Free nucleotides will affect the accuracy of quantification. Please purify RNA before using this method. The yield of RNA is determined by the A260 reading of the product. For single-stranded RNA, 1 A260 is equivalent to 40 µg/mL, so the yield of RNA can be calculated as follows: A260 x dilution factor x 40 = µg/mL RNA.

B. Dye method

Use RiboGreen dye to quantify RNA, free nucleotides won’t affect quantification, purified or unpurified RNA in reaction products can be accurately quantified.

5. RNA size and quality detection

A. Agarose electrophoresis

In order to determine the size, integrity and quality of RNA, agarose gel electrophoresis or polyacrylamide gel electrophoresis is required for detection.

B. Agilent Bioanalyzer detection

Agilent Bioanalyzer can be used to evaluate the integrity and quality of RNA. It only requires a small amount of RNA for analysis. High-quality RNA should show obvious and sharp peaks on the electrogram.

Notes:

Ø Be careful not to mix RNase in the reaction system.

Ø Experiment equipment (such as: pipette tip, product tube, etc.) should strictly use RNase-free products.

Ø For your safety and health, please wear lab coats and disposable gloves.

Ø For research use only!

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