Method for manufacturing single cell sequencing reference material by utilizing oral epithelial cells

A technique for oral epithelial cells and single-cell sequencing, applied in the field of molecular biology, can solve problems such as difficult to ensure the accuracy of isolated cells, and achieve the effects of large surface area, avoiding trauma, and easy identification

Inactive Publication Date: 2015-03-11
SUZHOU BASECARE MEDICAL DEVICE CO LTD
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Problems solved by technology

When separating nucleated cells from blood samples commonly used in clinical practice, they are usually disturbed by red blood cells, etc., and it is difficult to ensure the accuracy of cell separation

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  • Method for manufacturing single cell sequencing reference material by utilizing oral epithelial cells
  • Method for manufacturing single cell sequencing reference material by utilizing oral epithelial cells
  • Method for manufacturing single cell sequencing reference material by utilizing oral epithelial cells

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Embodiment Construction

[0028] The specific embodiments provided by the present invention will be described in detail below in conjunction with the accompanying drawings.

[0029] Single cells were obtained from the oral epithelial cells of 24 healthy persons, and 8 single-cell samples were obtained from each person's oral epithelial cells, including a negative control. Through methods such as electrophoresis analysis and comparison analysis of sequencing results, the feasibility of establishing human single cell-related experimental research standards by amplifying and sequencing single cells of human oral epithelial cells was confirmed.

[0030] 1. Acquisition and storage of human oral epithelial cells

[0031] Cell donors first rinse their mouths with cold boiled water to remove food residues in the mouth and ensure that the scraped cells do not contain other components other than human oral epithelial cells. Use a clean toothpick to flatten the cheeks on both sides of the mouth, scrape the oral ...

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Abstract

The invention relates to a method for manufacturing a single cell sequencing reference material by utilizing oral epithelial cells. The method comprises the following steps: (a) scrapping oral epithelial cells and saving in PBS (phosphate buffer solution); (b) sucking up a single oral epithelial cell by using capillary-opening suction pipe under a microscope; (c) amplifying a single-cell whole genome, and purifying the amplified single cell by using a paramagnetic particle method; (d) performing enzyme digestion on the amplified sample, stopping the experiment by using EDTA (ethylenediamine tetraacetic acid), and purifying by using the paramagnetic particle method; (e) constructing a Proton library; (f) performing computer sequencing on a Proton semiconductor high-throughput sequencing platform; (g) analyzing the sequencing data. The invention provides a new way of preparation of a standard product for single cell associated research; the method for manufacturing the single cell sequencing reference material by utilizing the oral epithelial cells has the characteristics that materials are simple to obtain, the operation is convenient and quick and the resolution is high, and is suitable for projects, such as PGS (preimplantation genetic screening) and tumor early screening.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for making a single-cell sequencing reference product using oral epithelial cells. Background technique [0002] With the improvement of gene sequencing technology and the successive development of major international cooperation projects such as the Thousand Genomes Project, Cancer Genome Project, and Meta-Hit Project, genome research has been pushed to a climax. However, the sequencing materials used so far have invariably been mixed DNA samples of millions or more cells. This method can obtain the whole genome sequence information, but the result obtained by studying it is only the average value of the signal in a group of cells, or only represents the information of the dominant number of cells, and the unique characteristics of a single cell are ignored. On the other hand, some samples are scarce and cannot be cultured in the laboratory, and the sample vo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 梁波孔令印申静静刘慧敏孙丹凤
Owner SUZHOU BASECARE MEDICAL DEVICE CO LTD
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