A method for detecting single cell 5-hmc

A detection method and single-cell technology, which is applied in the detection of modified bases and the field of analytical chemistry, can solve the problems of harsh chemical reaction conditions and low reaction efficiency, and achieve improved accuracy and sensitivity, simple reaction conditions, accurate detection and analysis Effect

Active Publication Date: 2021-09-07
XI AN JIAOTONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In order to overcome the shortcomings of the above-mentioned prior art, the object of the present invention is to provide a single-cell 5-hmC detection method, which is based on agarose gel microspheres wrapped with single cells, and realizes the detection of 5-hmC by using the click chemical reaction principle. Detection and analysis overcome the technical shortcomings of difficulty in detecting a large number of samples, inability to detect 5-hmC in a single cell, harsh chemical reaction conditions, and low reaction efficiency

Method used

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  • A method for detecting single cell 5-hmc
  • A method for detecting single cell 5-hmc
  • A method for detecting single cell 5-hmc

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Centrifuge the solidified single-cell gel microspheres at 3000rr for 2min and remove the supernatant; add isopropanol to the gelatin, shake and mix evenly, and centrifuge at 3000r for 2min to remove the supernatant; add absolute ethanol to the gelatin, After oscillating and mixing evenly, centrifuge at 3000r for 2min, remove the supernatant, and repeat this step twice; add PBS to the rubber balls, shake and mix evenly, and centrifuge at 3000r for 2min, remove the supernatant; complete the deemulsification of the rubber balls. Add 50μl 2×SSC to the lysed rubber balls, vortex and mix well, then centrifuge at 3000r for 2min, remove the supernatant; add 20μM SYBR-Green to the rubber balls, and shake in the DEPC water reaction system at 37°C and 500rpm in a dark environment React for 1 hour; after the reaction, centrifuge at 3000r for 2min and remove the supernatant; add PBS to the rubber balls, shake and mix well, and then centrifuge at 3000r for 2min. This step is repeated ...

Embodiment 2

[0044] Example 2: Comparison of experimental group and control group in MCF-7 cells and MCF-10A cells, and analysis and comparison of 5-hmC content in MCF-7 cells and MCF-10A cells.

[0045] Add 50μl NE Buffer 4 to the lysed gel microspheres, shake and mix well, centrifuge at 3000r for 2min, remove the supernatant; add 2μM β-GT and 200μM UDP-N3-Glu to the gel beads, and carry out in 1×NE Buffer4 Reaction, 37°C, 500rpm shaking reaction for 3h; after the reaction, centrifuge at 3000r for 2min, remove the supernatant; add 50μl PBS to the glue ball, shake and mix well, then centrifuge, remove the supernatant, repeat this step three times; add to the glue ball 50μM DBCO-Cy5, in 1×PBS solution, 37°C, 500rpm shaking reaction for 2h in a dark environment; after the reaction, centrifuge at 3000r for 2min, remove the supernatant; add 50μl 2×SSC to the rubber ball, shake and mix evenly, and then centrifuge at 3000r for 2min, Remove the supernatant; add 20 μM SYBR-Green to the gel balls, ...

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Abstract

The invention discloses a single-cell 5‑hmC detection method, which belongs to the technical field of molecular biology. The single-cell-wrapped agarose gel microspheres are obtained through a microfluidic chip method, and the single-cell-wrapped agarose gel microspheres are tested. After de-emulsification of the balls, the cells were lysed in the agarose gel microspheres, and then the Cy5 fluorescent group was connected to the genomic DNA by using the click chemical reaction principle to realize the chemical labeling of 5‑hmC on the genomic DNA. The invention utilizes the principle of click chemical reaction to realize the detection and analysis of 5-hmC, and overcomes the disadvantages of difficulty in detecting a large number of samples, inability to detect 5-hmC in a single cell, harsh chemical reaction conditions, and low reaction efficiency.

Description

technical field [0001] The invention belongs to the field of analytical chemistry and detection of modified bases, and relates to a single-cell 5-hmC detection method. Background technique [0002] Current studies have proved that the same kind of cells are significantly different from the microscopic point of view, that is, there are significant microscopic inconsistencies (heterogeneity). Traditional biological experiments are based on a large number of cells, and the experimental results are difficult to reflect the single-cell level. laws of life activity. Therefore, single-cell analysis can analyze the individual differences among cells of the same species, reveal the laws of life activities at a more microscopic level, and reveal the essence of life activities more accurately, and can also contribute to the exploration of the cause, development and treatment of major diseases. Provide more reliable scientific basis. Genomic DNA methylation is an important epigenetic ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6806C12N11/10
CPCC12N11/10C12Q1/6806C12Q2563/107C12Q2525/117C12Q2521/531
Inventor 赵永席张进薛静陈锋
Owner XI AN JIAOTONG UNIV
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