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Method for single cell detection and analysis and application thereof

A single-cell, cell technology, applied in the field of single-cell detection and analysis, can solve the problems of inability to achieve single-cell detection, small sample volume, and high detection sensitivity

Active Publication Date: 2021-05-11
BEIJING CENT FOR PHYSICAL & CHEM ANALYSIS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the qPCR in the prior art that depends on the Ct value, and requires a large amount of samples, which cannot meet the needs of rare cell detection. Although dPCR The demand for samples is small, but the detection of single cells cannot be realized. A method for detection and analysis of single cells is provided. This method has the advantages of small sample volume, high detection sensitivity, high accuracy, and can Accurately quantify the number of single cells, and at the same time detect the expression level of target proteins in rare cells at the single cell level, etc.

Method used

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  • Method for single cell detection and analysis and application thereof
  • Method for single cell detection and analysis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] This example is used to illustrate the effect of the single cell separation method provided by the present invention.

[0049] (1) Single cell isolation and verification of HepG2-GFP cells

[0050] In this example, HepG2 cells labeled with GFP were used for single cell isolation experiment. The construction method is as follows: the shuttle plasmid (PCDH-CMV-MCS-EF1-GFP-PURO, purchased from Youbao Biological Company) and the lentiviral packaging kit (Lenti-vpak packaging kit, purchased from OriGen×e Company, article number TR30037 ) in 293T cells (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) were co-transfected according to the instructions of the kit, at 37°C, 5% CO 2 After 48 hours of culturing under conditions, the virus liquid was collected (concentration was 10 8 / ml). The HepG2 cell suspension (concentration of 4 × 10 5 / ml) in a 6-well plate, the number of cells per well is about 4×10 5 , at 37°C, 5% CO 2 Cultivate overnight under conditi...

Embodiment 2

[0086] This example is used to illustrate the effect of the method provided by the present invention on the separation and detection of single cells at the expression level.

[0087] In this example, MCF-7A5 and MCF-7B10 cell lines were used for experiments. For the construction method, see Zeng Q, LiW, Lu D, et al. CD146, an epithelial-mesenchymal transition inducer, is associated with triple-negative breast cancer. Proc Natl Acad Sci U S A. 2012, 109(4): 1127-1132. These two cell lines were transformed from breast cancer cell MCF-7, which can stably express CD146. Among them, MCF-7A5 is a cell line with high expression of CD146, and MCF-7B10 is a cell line with moderate expression of CD146.

[0088] Prepare a single-cell separation system according to the ingredients and dosage in Table 4.

[0089] The reverse transcriptase used in Table 4 was purchased from Bio-Rad, Cat. No. 1864021. Its concentration is 40U / μL.

[0090] In the primer / probe Mix used in Table 4, the final...

Embodiment 3

[0105] This embodiment is used to illustrate the impact of the concentration of SDS and additives in the system on the accuracy of the method provided by the present invention.

[0106] (1) Effect of SDS concentration in the system on single cell separation and detection results

[0107] The single cell separation experiment was carried out using the method in Example 1 (1), the difference being that the number of cells in the system was fixed at 1000, and the final concentrations of SDS were respectively 0, 0.001 wt%, 0.01 wt%, and 0.15 wt%. and 1.5% by weight. Experiments were carried out according to the single-cell separation system in Table 1. The total volume of the system was 20 μL, and the different concentration gradients of SDS were realized by adjusting the addition of SDS solution and water.

[0108] The number of positive droplets and total droplets in the system containing different concentrations of SDS was analyzed, and the results showed that: as Figure 7 A...

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Abstract

The invention relates to the field of single cell detection and analysis, and discloses a method for single cell detection and analysis. According to the method, single cell separation and cell lysis are carried out on a sample by a one-step method by utilizing a digital PCR instrument, and a target sequence in the sample is detected and analyzed, so that the application range of the digital PCR instrument is expanded, and the digital PCR instrument can be used for detecting micro cells; and meanwhile, a target gene in each single cell in the sample can be accurately quantified, the detection of the expression level of the target gene can be realized, and the method can also be used for verifying a single cell sequencing result.

Description

technical field [0001] The invention relates to the field of single cell detection and analysis, in particular to a method for single cell detection and analysis and its application. Background technique [0002] Single-cell analysis technology is a new type of cell research technology with high sensitivity, high selectivity, and high spatiotemporal resolution. Currently, common single-cell analysis technologies include single-cell separation and single-cell sequencing. Single-cell analysis technology is very suitable for biological and medical detection due to its high sensitivity and accuracy. [0003] Digital PCR (dPCR) technology is a technology based on microfluidics for the absolute quantification of nucleic acid molecules. This technology divides the sample into microdroplets, distributes the target nucleic acid molecules in the microdroplets, and distributes the target nucleic acid molecules in each microdroplet. The number of target nucleic acid molecules is 0-1, a...

Claims

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Application Information

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IPC IPC(8): C12Q1/686C12Q1/6858
CPCC12Q1/686C12Q1/6858C12Q2527/125C12Q2563/159C12Q2531/113
Inventor 安云鹤程小艳涂晨晓张小莉洪甜杨双田赛
Owner BEIJING CENT FOR PHYSICAL & CHEM ANALYSIS
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