Method for detecting RNA m6A with high flux, high sensitivity and single base resolution and application thereof

A high-sensitivity, high-throughput technology, applied in the field of high-throughput and high-sensitivity single-base resolution detection of RNAm6A, can solve the problems of cumbersome miCLIP process, difficult to achieve m6A detection, and high experimental conditions, and achieve antibody enrichment efficiency. The effect of improving, improving sensitivity, and reducing false positive rate

Pending Publication Date: 2020-08-21
SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although both meRIP and miCLIP can detect m6A information at the whole transcriptome level, there are still some shortcomings: 1. These two methods require a large amount of sample RNA, and the general amount of RNA is about 500ug. It is difficult to realize the detection of m6A in the medium; second, meRIP is realized by fragmenting the RNA and enriching it according to the m6A antibody, and the final result is a fragment with m6A sites enriched by the antibody, and the resolution is low; 3. miCLIP only determines the mutation site through the peaks obtained by antibody enrichment, and there will be a high false positive rate for RNA with a high background expression level; 4. The process of miCLIP is cumbersome, with many steps, and requirements for experimental conditions higher

Method used

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  • Method for detecting RNA m6A with high flux, high sensitivity and single base resolution and application thereof
  • Method for detecting RNA m6A with high flux, high sensitivity and single base resolution and application thereof
  • Method for detecting RNA m6A with high flux, high sensitivity and single base resolution and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] The application of hmCLIP in fine-needle aspiration paraffin specimens of thyroid cancer involves the following steps in sequence (flow chart as shown in figure 1 shown):

[0073] 1. Obtain total RNA

[0074] Three to five fine-needle aspiration paraffin specimens of thyroid cancer were taken, and total RNA was extracted with QIAGEN RNeasy FFPE kit. The specific method is as follows:

[0075] 1) Dewaxing of paraffin sections: take 3 to 5 paraffin sections, depending on the size of the section tissue, put them in a 1.5ml nuclease-free EP tube, add 1ml xylene, vortex fully for 10s, and centrifuge at the maximum speed for 2min;

[0076] 2) Remove the supernatant as much as possible, add 1ml of absolute ethanol to the precipitate, vortex to mix, and centrifuge at the maximum speed for 2min;

[0077] 3) Remove the supernatant as much as possible, open the lid and place at room temperature for 10 minutes until the residual ethanol evaporates;

[0078] 4) Add 150ul PKD buff...

Embodiment 2

[0269] Referring to steps 1 to 5 in Example 1, the difference is that the cross-linking energy and time of RNA and m6A antibody in step 4 (co-immunoprecipitation and cross-linking) are different, and are divided into the following 6 groups of experiments:

[0270] (1) No UV crosslinking (named UV-);

[0271] (2) Use Spectroline UV 254nm Instrument (model: XLE-1000) for cross-linking, cross-linking energy: 150mj / cm 2, Number of cross-links: 2 times (named: 150mj / cm 2 *2);

[0272] (3) Use Spectroline UV 254nm Instrument (model: XLE-1000) for cross-linking, cross-linking energy: 150mj / cm 2 , Number of crosslinks: 4 times (named: 150mj / cm 2 *4);

[0273] (4) Use Spectroline UV 254nm Instrument (model: XLE-1000) for cross-linking, cross-linking energy: 300mj / cm 2 , Number of cross-links: 2 times (named: 300mj / cm 2 *2);

[0274] (5) Use Spectroline UV 254nm Instrument (model: XLE-1000) for cross-linking, cross-linking energy: 400mj / cm 2 , Number of crosslinks: 2 times (...

Embodiment 3

[0278] The method is the same as that of Example 1 (steps 1 to 5.9), the difference is: the concentration of RNA Ligase High conc when the RNA adapter is connected in step 5, the 3' end is connected to the RNA adapter in step 6, and the 3' adapter of cDNA in step 7 is built. For high concentration ligase (T4 RNA Ligase 1, High Concentration (NEB: M0437M)) and common ligase (T4 RNA Ligase, NEB: M0204S) (initial concentrations are 30,000U / ml and 10,000U / ml respectively), Its ligation efficiency as well as the reproducibility of hmCLIP such as Figure 8 Shown (reproducibility of hmCLIP is a repeat of Examples 1 to 5.9)).

[0279] From Figure 8 It can be seen from the figure that the signal of the ligation product obtained by using a high concentration of ligase to connect the fluorescently-labeled adapter is significantly higher than that of the common ligase, and the reproducibility is also high.

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Abstract

The invention discloses a method for detecting RNA m6A with high flux, high sensitivity and single base resolution and an application thereof. The method comprises the following steps: (1) extractingtotal RNA in a sample and removing rRNA to obtain an RNA sample; (2) fragmenting the RNA sample, and dividing the fragmented RNA sample into two parts, namely an RNA fragment I and an RNA fragment II;(3) carrying out co-immunoprecipitation and cross-linking treatment on the RNA fragment I; (4) respectively carrying out 3'tail end joint connection on the complex obtained by cross-linking treatmentand the RNA fragment II; (5) respectively carrying out reverse transcription to obtain cDNA, connecting the cDNA with a 3'end joint, carrying out PCR amplification, and respectively constructing an hmCLIP library and an input library; and (6) sequencing the hmCLIP library and the input library by using a high-throughput sequencing platform. The method is short in consumed time, high in sensitivity and suitable for detecting RNA m6A in a small number of specimens and specimens with low RNA integrity.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for detecting RNAm6A with high-throughput and high-sensitivity single-base resolution and its application. Background technique [0002] The m6A modification of RNA occurs at the N6 position of adenine nucleotide in RNA, which is the most common type of apparent post-transcriptional modification on eukaryotic mRNA, accounting for about 0.1-0.4% of adenine on mRNA. The m6A modification process of RNA is catalyzed by the methyltransferase complex and demethylated by the demethyltransferase, so it is a dynamically reversible process. More and more studies have shown that m6A plays an important role in important life processes such as embryonic development, neurogenesis, tumor progression, and sex determination. Therefore, it is extremely important to accurately locate the m6A modification site of RNA to study the specific mechanism of m6A regulating life activities. However, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2525/191C12Q2535/122C12Q2563/143C12Q2563/149
Inventor 尹东汪颜杰廖建友
Owner SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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