Method for detecting 5-methylcystein in RNA and kit thereof

A technology of hydroxymethylcytosine and detection kits, applied in the field of biochemistry, can solve the problems of high cost, cumbersome operation, and long time consumption, and achieve the effects of easy operation, simple sample preparation, and high sensitivity

Active Publication Date: 2016-08-24
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this detection method is time-consuming, expensive, inconvenient, cumbersome to operate, and troublesome to prepare samples.

Method used

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  • Method for detecting 5-methylcystein in RNA and kit thereof
  • Method for detecting 5-methylcystein in RNA and kit thereof
  • Method for detecting 5-methylcystein in RNA and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1 RNA 5hmC detection method

[0076] 1. Extract total RNA and dilute to a concentration of 100ng / μL.

[0077] 2. Add an equal volume of 2M NaOH solution and react at 4°C for 30 minutes.

[0078] 3. Take a 4×8 nitrocellulose membrane, drop 2 μL of samples onto the membrane sequentially, and place it at room temperature for 15 minutes.

[0079] 4. Place the film in an oven at 80°C and bake for 30 minutes.

[0080] 5. After taking it out from 80°C, block it with 5% milk, react at room temperature for 1 hour, and then wash it with PBST solution 3 times, 10 minutes each time.

[0081] 6. Incubate the NC membrane with the 5hmC antibody diluted in PBS solution, react overnight at 4°C, and then wash with PBST solution 3 times, 10 minutes each time.

[0082] 7. Incubate the NC membrane with a specific secondary antibody diluted with 5% milk, react at room temperature for 1 hour, and then wash with PBST solution 3 times, 10 minutes each time.

[0083] 8. After develop...

Embodiment 2

[0086] Example 2 Effects of Different Denaturing Agents and Concentrations on RNA 5hmC Detection

[0087] (1) The influence of different denaturing reagents on the detection effect of RNA 5hmC

[0088] Denaturing reagent type setting: NaOH, formaldehyde, Tris-HCl;

[0089] The test method is:

[0090] 1. Extract total RNA and dilute to a concentration of 100ng / μL.

[0091] 2. Add an equal volume of denaturing reagent solution (NaOH, formaldehyde or Tris-HCl) with a concentration of 2M, and react at 4°C for 30 minutes.

[0092] 3. Take a 4×8 nitrocellulose membrane, drop 2 μL of samples onto the membrane sequentially, and place it at room temperature for 15 minutes.

[0093] 4. Place the film in an oven at 80°C and bake for 30 minutes.

[0094] 5. After taking it out from 80°C, block it with 5% milk, react at room temperature for 1 hour, and then wash it with PBST solution 3 times, 10 minutes each time.

[0095] 6. Incubate the NC membrane with the 5hmC antibody diluted in...

Embodiment 3

[0115] Example 3 Effects of different denaturation times on the detection effect of RNA 5hmC

[0116] Denaturation time setting: 30min, 1h, 3h, 6h, 12h, 24h, 48h;

[0117] The test method is:

[0118] 1. Extract total RNA and dilute to a concentration of 100ng / μL.

[0119] 2. Add an equal volume of NaOH with a concentration of 2M, and react at 4°C for a certain period of time (respectively 30min, 1h, 3h, 6h, 12h, 24h, 48h).

[0120] 3. Take a 4×8 nitrocellulose membrane, drop 2 μL of samples onto the membrane sequentially, and place it at room temperature for 15 minutes.

[0121] 4. Place the film in an oven at 80°C and bake for 30 minutes.

[0122] 5. After taking it out from 80°C, block it with 5% milk, react at room temperature for 1 hour, and then wash it with PBST solution 3 times, 10 minutes each time.

[0123] 6. Incubate the NC membrane with the 5hmC antibody diluted in PBS solution, react overnight at 4°C, and then wash with PBST solution 3 times, 10 minutes each ...

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PUM

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Abstract

The invention relates to the technical field of biochemistry, in particular to a method for detecting 5-methylcystein in RNA and a kit thereof. The method for detecting 5-methylcystein in RNA comprises the steps that total RNA and a modified reagent are mixed and modified, and modified RNA is obtained; the modified RNA is combined on a nitrocellulose membrane, drying and sealing are carried out, and an antigen membrane is obtained; the antigen membrane and a 5-hydroxymethylcytosine antibody are subjected to first incubation, then the antigen membrane and a second specific antibody are subjected to second incubation, development is carried out, and a detecting result is obtained. According to the detecting method, 5-methylcystein in RNA can be detected and analyzed fast, high sensitivity is achieved, operation is easy, and sample preparation is easy.

Description

technical field [0001] The invention relates to the technical field of biochemistry, in particular to a method for detecting 5-hydroxymethylcytosine in RNA and a kit thereof. Background technique [0002] 5-Hydroxymethylcytosine (5hmC) was first discovered in phage DNA in 1952. It can be modified by glycosyltransferase-mediated glycosylation, so that the phage genome can resist the degradation of host restriction enzymes after entering the host. . In 1972, Penn et al. also found hydroxylated cytosine in the DNA extracted from the brain tissue of mammalian rats, mice, and oviparous bullfrogs, accounting for about 15% of the total cytosine in DNA, but subsequent studies have not been repeated. This result. It was not until 2009 that the two research teams of Tahiliani and Kriaucionis reported in the same issue of Science that 5hmC was abundantly expressed in human, mouse brain and embryonic stem cells, and the concept of hydroxymethylation came into people's field of vision ...

Claims

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Application Information

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IPC IPC(8): G01N33/544
CPCG01N33/544
Inventor 徐兴顺苗志刚
Owner SUZHOU UNIV
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