Method for cell quality control through unicellular transcriptome sequencing

A transcriptome sequencing and single-cell technology, applied in the field of molecular biology, can solve problems such as no effective control of cell quality, and achieve better therapeutic effects and better quality control results

Active Publication Date: 2017-05-24
GENEIS TECH BEIJING CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] To sum up, there is no technology for effectively controlling the quality of cells in the prior art

Method used

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  • Method for cell quality control through unicellular transcriptome sequencing
  • Method for cell quality control through unicellular transcriptome sequencing
  • Method for cell quality control through unicellular transcriptome sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Sample preparation

[0065]After the collected cells were resuspended and mixed evenly, they were counted. The counting instrument used: count star. The specific method is: mix fuel and cells 1:1, draw 20 μl of the mixture and put it into the counting plate, then put it into the instrument, calculate the cell density and count the ratio of cell particle size, usually control the cell density at 166-250K / mL Among them, choose the model of Fluidigm's C1 single-cell capture chip according to the size of the cell particle.

Embodiment 2

[0067] ChIP Primer

[0068] According to the Fluidigm operating instructions, add the corresponding reagents to the corresponding positions of the chip, then put it into the C1 single cell capture instrument, and select the program mRNA seq: prime to pretreat the chip for about 10-15 minutes.

Embodiment 3

[0070] Load cells

[0071] Mix the prepared cells with the suspension reagent provided by Fluidigm, the system is as follows:

[0072] Cells 166-250K / mL 60μL C1cells suspension Reagent (Fluidigm) 40μL total capacity 100μL

[0073] According to the Fluidigm operating instructions, add the corresponding reagents and prepared cells to the corresponding positions of the chip, then put them on the C1 single cell capture instrument, select the program mRNA Seq: Cell Load, and perform single cell capture. This process takes about 30 minutes. After the program is over, take the chip out of the instrument, observe it under a microscope, and record which of the wells 1-99 are single cells.

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Abstract

The invention provides a method for cell quality control through unicellular transcriptome sequencing. The method comprises the following steps of: performing unicellular separation so as to obtain a total unicellular number; performing transcriptome amplification on each unicell; confirming that amplification can be implemented but the unicellular number is not sufficient for analysis on sequencing results; performing sequencing and analysis, including performing main component analysis and cluster analysis on the sequencing results, comparing with a standard unicellular database, and calculating the unicellular number off from total tendency; screening, including screening based on unicellular partitioning and screening based on expression gene types, wherein the step of screening based on unicellular partitioning comprises a step of screening cells of which the activity levels are greater than 75%, and the step of screening based on the expression gene types comprises a step of classifying expression genes into a good gene group, a neutral gene group and a malignant gene group according to gene functions. By adopting the method provided by the invention, the uniformity or the homogeneity of cells can be effectively controlled.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for cell quality control through single-cell transcriptome sequencing. Background technique [0002] Cell therapy refers to using the characteristics of certain cells with specific functions, using bioengineering methods to obtain and / or through in vitro expansion, special culture, etc., to make these cells have the ability to enhance immunity, kill pathogens and tumor cells, and promote tissue regeneration. Therapeutic effects such as organ regeneration and body rehabilitation, so as to achieve the purpose of treating diseases. The key process of cell therapy is to obtain cells, expand them and infuse them back into the patient's body. Therefore, the safety of cells is a very important issue to consider. [0003] There are few published reports on the quality control of cells in cell therapy. The limited public literature only involves safety issues such as whether th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/02G06F19/24
CPCC12Q1/6869G16B40/00C12Q2531/113
Inventor 田埂郎继东张丽娜
Owner GENEIS TECH BEIJING CO LTD
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