Method for rapidly and quantitatively detecting capping efficiency of RNA

A technology for RNA capping and quantitative detection, which is applied in the field of RNA synthesis research and can solve problems such as complex operations

Pending Publication Date: 2021-04-09
HUAZHONG UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although these two methods have the advantage of quantitatively measuring the RNA capping efficiency compared to the isotope method, the operation is still relatively complicated

Method used

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  • Method for rapidly and quantitatively detecting capping efficiency of RNA
  • Method for rapidly and quantitatively detecting capping efficiency of RNA
  • Method for rapidly and quantitatively detecting capping efficiency of RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] In this example, vaccinia virus capping enzyme and co-transcription method were used to cap sox7 RNA respectively, and then the calf intestinal alkaline phosphatase (CIP, #M0290) produced by NEB was used to dephosphorylate the RNA, and the RNA After purification, use T4 polynucleotide kinase (PNK, #M0201) produced by NEB to add monophosphate to the 5' end of RNA to obtain monophosphate RNA, and then use monophosphatase produced by NEB (XRN-1, #M0338 ) to digest monophosphorylated RNA, and finally run the gel to detect and quantitatively measure the brightness of the band, thereby distinguishing capped and uncapped RNA, and achieving the purpose of rapid quantitative detection of RNA capping efficiency. The specific steps are as follows:

[0031] S1. Synthesis of uncapped RNA by in vitro transcription: Using purified linearized DNA as a transcription template, KP34 RNA polymerase binds to a specific promoter on the template and initiates in vitro transcription of RNA to o...

Embodiment 2

[0057] The differences between this example and Example 1 are: (1) In this example, cas9 RNA is used for the reaction. (2) In steps S1 and S2 of Example 1, the KP34 RNA polymerase in the transcription and co-transcription reaction system was replaced with VSW3 RNA polymerase, and the transcription reaction was carried out at 16° C., and the transcription time was 16 hours.

[0058] The results of final gel running results and RNA capping efficiency are shown in the attached figure 2 shown.

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Abstract

The invention discloses a method for rapidly and quantitatively detecting the capping efficiency of RNA. The method comprises the following steps: S1, performing in-vitro transcription to synthesize uncapped RNA and removing template DNA; S2, carrying out capping treatment on the RNA; S3, performing mono-phosphorylation treatment on the RNA obtained in the steps S1 and S2: firstly, performing dephosphorylation by using alkaline phosphatase, purifying the RNA, and then adding monophosphate to the 5' end of the RNA by using polynucleotide kinase; S4, removing the RNA subjected to the monophosphorylation treatment by using monophosphatase, and setting a control group not treated with the monophosphatase; and S5, performing glue leaking detection: quantitatively determining the strip brightness (denoted as n) of the RNA treated by the monophosphatase and the strip brightness (denoted as N) of the RNA of the control group, and calculating the capping efficiency of the RNA: (n / N)*100%. The method has the beneficial effects that (1) the capping efficiency of the RNA can be rapidly and quantitatively detected, the operation is simple and rapid, and the result is effective and accurate; and (2) the method has less demand on samples, has no special requirements on RNA types, lengths and the like, and has a wide application range.

Description

technical field [0001] The invention belongs to the field of RNA synthesis research, in particular to a method for rapidly and quantitatively detecting RNA capping efficiency. Background technique [0002] RNA (ribonucleic acid) is an important macromolecule in the transmission of all biological genetic information, and it also plays a variety of regulatory roles in organisms. In recent years, RNA research has become one of the most popular fields in biological research. For example, RNA, as a drug, has much fewer side effects than gene therapy, a method that changes DNA to produce permanent and irreversible effects. Therefore, RNA therapy has gradually become the most popular drug research direction, and it is expected to become an effective method for many diseases. Good development prospects. [0003] Effective mRNA therapy requires efficient delivery of mRNA to patients and efficient synthesis of corresponding proteins in vivo. To optimize mRNA delivery and protein syn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6809
CPCC12Q1/6809
Inventor 朱斌成锐夏恒黄锋涛吴慧陆雪玲闫艳余兵兵王雄略蒋怡心
Owner HUAZHONG UNIV OF SCI & TECH
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