Product Description

T7 High Yield RNA Synthesis Kit optimizes the transcription reaction system. The kit can synthesize the single-stranded RNA efficiently by uses T7 RNA polymerase, the linear double-stranded DNA with the T7 promoter sequence as the template, NTPs as the substrate to control the DNA sequence downstream of the promoter. During transcription, modified nucleotides can be added to the substrate to prepare biotin or dye-labeled RNA.

This kit can synthesize long transcripts and short transcripts, RNA can be produced 100-200 μg with 1 μg of DNA template input. The RNA synthesized by transcription can be used for various downstream applications, such as RNA structure and function research, RNase protection, probe hybridization, RNAi, microinjection and in vitro translation.

 

Contents

Contents No.	Name	Cat#/Specification
		10623ES50 (50 T)	10623ES60 (100 T)	10623ES70 (500 T)
10623-A	T7 RNA Polymerase Mix	100 μL	200 μL	1 mL
10623-B	10×Transcription Buffer	100 μL	200 μL	1 mL
10623-C	ATP（100mM）	100 μL	200 μL	1 mL
10623-D	CTP（100mM）	100 μL	200 μL	1 mL
10623-E	GTP（100mM）	100 μL	200 μL	1 mL
10623-F	UTP（100mM）	100 μL	200 μL	1 mL
10623-G	Control DNA Template（500ng/μL）	10 μL	20 μL	100μL

 

Applications

In vitro RNA synthesis

 

Shipping and Storage

Dry ice transportation. Store at -20℃, valid for two years.

 

Notes:

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

2. Experiment equipment (such as: pipette tip, product tube, etc.) should strictly use RNase Free products.

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

4. For research use only!

 

Synthesis principle

 

Figure 1: In vitro RNA transcription process

 

Experimental methods

1. DNA template preparation

Linearized plasmids with double-stranded T7 promoters or PCR amplification products can be used as Hifair^TM T7 High Yield RNA Synthesis Kit in vitro transcription templates, which can be dissolved in TE buffer or RNase free H₂O.

T7 promoter sequence: TAATACGACTCACTATAG*GG (Note: G* 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.
Note: 1. 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.
2. 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.
3. 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 CTCACTATAGGG) 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.
Note: 1. 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.
2. 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 transcription
A. Thawing reagents

Centrifuge the T7 RNA Polymerase Mix briefly and place on ice. Thaw 10× Transcription Buffer and ribonucleotides (ATP, CTP, GTP, UTP), mix and centrifuge to the bottom of the tube, place 10× Transcription Buffer at room temperature, and place 4 types of ribonucleotides on ice.
B. Assembly transcription reaction at room temperature

Prepare the reaction system according to the following system:

 

Components	Volume（μL）	Final concentration
RNase free H2O	Up to 20	–
10×Transcription Buffer	2	1×
CTP / GTP/ ATP/ UTP (100 mM each)	2 each	10 mM each
Template DNA	1 µg	–
T7 RNA Polymerase Mix	2	–

Note: 1. 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.
2. Short transcript (<100 nt), 2 µg template can be used, transcription time increased to 4-8 hs.
3. For long transcripts (>1000 nt), recommended to use linearized plasmid templates for transcription.
4. Perform the reaction in a PCR machine with the hot lid open to prevent the reaction solution from evaporating for a long time.
5. 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.
6. The reagents and containers must without RNase contamination.
C. Incubate at 37°C for 2 hours

Mix the above reaction solution, briefly centrifuge to the bottom of the tube, and incubate at 37°C for 2 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 2 μL of DNase I (RNase free) to each tube and incubate at 37°C for 15 mins to remove the template DNA.

3. Product purification

A. RNA Cleaner magnetic bead

Take out RNA clean beads from 4°C in advance, equilibrate to room temperature (about 30 minutes), dilute the transcription product to 50 μL with RNase free H₂O.

①Invert or vortex to mix the magnetic beads thoroughly, add 2× magnetic beads (100 μL) to the RNA sample (50 μL), pipette 6 times to mix thoroughly. Incubate for 5 min at room temperature, allow RNA to bind to the magnetic beads.

②Place the sample on the magnetic stand for 5 mins. After the solution is clear, remove the supernatant carefully.

③Keep the sample on the magnetic stand, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 s, carefully remove the supernatant. Repeat this operation once again.

(Note: The 80% ethanol used for rinsing needs to be freshly prepared with RNase free H₂O to prevent the introduction of RNase enzyme from causing RNA degradation.)

④Keep the sample on the magnetic stand, open the lid for 5 mins to dry the beads.

(Note: Avoid excessive drying. If the magnetic beads are cracked, it means that the magnetic beads are too dry and the elution efficiency of RNA will be reduced).

⑤Remove the sample from the magnetic stand, add 22 μL RNase free H₂O, pipette 6 times to mix well, incubate for 5 mins at room temperature.

⑥Place the sample on the magnetic stand for 5 mins. After the solution clarifies, carefully transfer 20 μL of the supernatant to a new RNase free PCR tube.

Note: Leave 2-3 μL of liquid when transferring the supernatant, not attract the magnetic beads and affect the subsequent experiments. The obtained RNA is extremely unstable, please proceed to the next step as soon as possible. If you want to save, please store at -80°C.)

B. Phenol/chloroform

①Add 115μL RNase free H₂O and 15 μL 3M sodium acetate (pH 5.2) to 20 μL reaction mixture, mix well.

②Extract once with an equal volume of phenol/chloroform (1:1), and then extract twice with an equal volume of chloroform. Collect the supernatant and transfer it to a new RNase free EP tube.

③Add 2 volumes of absolute ethanol to precipitate RNA. Mixing uniformly and place it at -20°C for at least 30 mins, centrifuge at 4°C for 15 mins at maximum speed, and collect the precipitate.

④Add 500 μL of ice-cold 70% ethanol to wash the RNA pellet.

⑤Dissolve the RNA pellet with 20 μL RNase free H₂O. Store the purified RNA solution at -80°C.

C. Lithium chloride precipitation

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 7.5M 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.

D. Column purification

Before purification, add 80μL RNase free H₂O to dilute the product to 100 μL, and then follow the column purification instructions for purification.

4. RNA quantification

A. Ultraviolet absorption

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 2100 Bioanalyzer detection

Agilent 2100 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.

 

Frequently Asked Questions: 

1. Low transcript yield

The quality of template is closely related to the yield. The yield of experimental group is significantly lower than the control group. The possible reasons are: ① the experimental template contains inhibitory components; ② The template has something wrong.

Suggestions: ① Re-purify the template; ② Determine the template quantification and its integrity; ③ Extend the reaction time; ④ Increase the amount of template input; ⑤ Try other promoters and RNA polymerases.

2. Low yield of short transcripts

Short transcription initiation fragment will inhibit the reaction. When the transcription product is less than 100 nt, extending the reaction time to 4-8 hs or increasing the amount of template to 2 μg will increase RNA yield.

3. RNA transcription length is greater than expected

If the electrophoresis shows that the product band is larger than the expected size, the possible reasons: ①The plasmid template may not be completely linearized; ②The 3' end of the sense strand has a prominent structure; ③The RNA has a secondary structure that is not completely denatured.

Suggestions: ①Check whether the template is completely linearized, and if necessary, perform additional linearization; ②Select a suitable restriction enzyme to avoid 3' overhangs, or use Klenow Fragment /T4 DNA polymerase to complete the transcription before proceeding; ③Use denatured gel to detect RNA products.

4. RNA transcription length is less than expected

If the electrophoresis shows that the product band is smaller than the expected size, the possible reasons: ①The template contains a termination sequence similar to T7 RNA polymerase; ②The GC content in the template is high.

Suggestions: ①Lower the reaction temperature (for example, 30°C). Sometimes lowering the temperature can increase the transcription length, but it will reduce the yield. Or try different RNA polymerases for transcription; ②If the template GC content is high, use 42℃ to transcript, or add SSB to increase the yield and transcription length.

5. Electrophoresis tailing of transcription products

There is tailing phenomenon during electrophoresis. Possible reasons: ①Contaminated by RNase during experimental operation; ②Contaminated DNA template by RNase.

Suggestions: ①Use RNase-free pipette tips and EP tubes, wear disposable latex gloves and masks, and all reagents are prepared with RNase free H₂O. ②Re-purify the template DNA.

HB220514

Q：Control DNA Template 的长度是多少？

A：大小是1142bp。

Q：T7 试剂盒有帽子结构和polyA 吗?

A：polyA 尾是设计模板时引入，加帽是单独加。

Q: IVT体系如何优化？

A: ①提高NTP，帽类似物的投入量比例；②添加剂的合理应用；③反应温度可以用到40度。

IVT体系优化可以从模板，T7酶的使用量，如果是共转录加帽可以考虑帽子与NTP之间的比例；酶法加帽的话，在加帽酶，2-O-甲基转移酶以及SAM的用量配比上进行条件优化。关于模板除了线性化要完全这一块，还有一点就是序列设计这一块，例如在5’UTR区注意uAUG以及Kozak序列；CDS需要考虑密码子偏好性，GC3含量，双核苷酸偏好性等；3’UTR：RBP以及miRNA结合位点：还有一个就是RNA的二级结构，5’UTR前序列稳定的二级结构会降低mRNA的表达；3’UTR稳定的二级结构会提高mRNA稳定性，促进mRNA编码蛋白的表达。推荐的IVT体系和更多细节tips，请参考金畔生物mRNA体外合成实验手册。

Q: IVT反应20ul体系放大到10ml,产量下降，单位体积产物得率下降约50%；你们是否遇到过这种情况，有何建议？

A: 20uL体系等比放大到10mL应该得率差不多的，我们一般从20uL直接放大到50mL，得率基本也在100ug/20uL的水平；我们50mL也会用离心管和金属浴的方式来测，10mL正常来说应该放大效果更好；这种情况下，一般建议酶和NTP的终浓度微调一下再试试。

Q: 现在大多数mRNA完整性能做到多少？

A: 就我们的其他客户返样，我们用Qsep100测的CE结果来看，比较理想的状况CE完整度能到88~92％；当然做到这个水平通常也需要经过一系列的优化；

一般产量是在120-220ug/ul（产量与序列设计有很大关系），共转录方式，mRNA产量有8g/L，做工艺优化产量会提升，目前市面上的工艺质量参数，有的是观察GFP的荧光值，有的是用lc-MS测量加帽率。

Q: 你们自己测试过程中是否会用琼脂糖电泳来检测mRNA产物？

A：RNA电泳我们也会做，主要是简要的看一下产物纯度，是否有明显的聚体这些；如果是看RNA大小，电泳的时候一般会点一个明确大小的RNA样，作为参考。

Q: 你们会对DNA模板中的Rnase残留进行检测吗？

A: 对模板的检测目前能找到的主要是序列完整性还有内毒素、宿主核酸残留这些检项，RNase还没有明确找到对应的检测建议方法；或者也可以参考我们酶产品中RNase残留的检测方法，相对还是比较简单可靠的。

Q: IVT反应过程中dsRNA的生成机制是什么？

A：IVT过程中的ds生成有多种可能的原因，主要有：

①T7 pol会以产物RNA作为模板，在其3'末端继续延伸RNA，延长序列与原RNA分子3'末端序列互补配对，形成发卡结构；

②T7 pol可以把RNA当作模板来复制RNA分子，合成与其互补的另外一条链；

（除去dsRNA目前是没有很好的方式，有专利报导用纤维柱（sigma）可以试一试）

Q: 控制体外转录阶段产物中dsRNA含量是否有可行的方案？

A：关于优化IVT体系与反应条件抑制dsRNA的建议主要还是以下几点：

①降低镁离子浓度；但降低镁离子浓度也会导致RNA合成产量的降低，需要去一个平衡点；

②提高IVT反应温度，50-56℃的反应温度能有效降低产物中dsRNA的含量；但是高温反应对工艺放大设备的要求会不一样，也可能提高成本；

③IVT反应体系中添加某些离散剂，如尿素、甲酰胺等；这个路线我们的工艺研发团队也做了一些摸索，是可以起到抑制dsRNA的效果的，但是否适合工艺放大还有待进一步研究

Q: IVT的反应过程中出现沉淀的原因是什么，怎么优化？

A: 内部有研究过这个沉淀，是镁离子、RNA、钠离子和焦磷酸镁的聚合物，且有个凝聚作用，聚团会不断变大（类似的胶状、絮状、颗粒沉淀都是类似原理）， 加水和加EDTA的效果类似也佐证了这个说法。原因：1.Mg离子浓度过高会和焦磷酸聚集形成焦磷酸镁沉淀，但是具有聚合作用，不断地包裹核酸形成胶状物导致产量下降。2.RNA产量较高也会析出沉淀。

这种沉淀的产生是偶然性的，与序列设计也有很大关系。

解决办法：（1）减少镁离子的浓度，但会牺牲产量，需要增加T7酶用量以及延长反应时间（从2h，延长到3-4h）;（2）NTP的存在形式（镁离子浓度高，例如46mM是不会挑NTP的，但当镁离子浓度降低，NTP（Tris盐形式）产量无太大变化，三Na形式的NTP产量下降明显。

Q: mRNA生产阶段工艺放大的反应器选择有什么思路？

A：可以考虑WAVE、海道夫反应釜、赛多利斯，主要还是需要找到和现有IVT体系适配的袋子；对于搅拌罐体，螺旋桨的转速也需要根据实际情况优化，跟桨叶类型和大小有关系，建议可以向设备生产厂家咨询。

Q: 你们推荐两步法的还是一步法的？为啥目前很多都还是使用的两步法的？

A: 工业上酶法加帽最常使用的是牛痘病毒加帽酶处理IVT产物可以将其修饰成Cap 0 mRNA，Cap 0结构可以在二氧甲基转移酶（2'O-methyltransferase）的作用下进一步修饰成Cap 1（m7GpppmN）。利用酶法加帽，加帽效率可以达到95%以上。共转录加帽法操作简便，但由于GTP会竞争帽状二聚体，因此该方法加帽率低一些；两种方式各有优缺点。 

Q: IVT体外转录产量有多少？
A: 目前我司体系为20ul的反应体系，体外转录的产量为100-200ug，内部的放大体系10ml可达到50mg—100mg。

Q: 体外转录随着反应时间的延长大于2h之后，完整度和产量比较会降低？生成的mRNA如何保证稳定性？

A: 建议整个反应时间在2-3小时之间，我司验证最长3h反应，合作客户有验证4h  反应结果正常。

Q: 是否可以帮忙设计mRNA体外转录DNA模板序列（主要是新冠疫苗应用方向的）？

A: 不提供相关服务。新冠疫苗目前主要可以直接参考的还是外网开源的BioNtech和Moderna的疫苗序列。主要原件序列基本是可靠的，除了polyA尾不全；国内比较早的新冠疫苗，还是基于RBD蛋白序列的比较多。这块我们没有要到具体的序列，基本都是各个研发团队的核心技术了。

Q: 翌圣加帽率、加尾检测用的什么方法，什么仪器？

A: 加帽用的是安捷伦系列（6230B TOF），加尾用的Thermo的QE或waters的Rda。

Q: 你们加A尾的方式是采用加尾酶的吗？你们一般建议连接多少个碱基？

A: 我们这个试剂盒没有加poly A尾的组分，可以在模板制备阶段将A尾构建到质粒上，长度一般在20-200。

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