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Single cell lysis buffer and application thereof

A lysate, single-cell technology, applied in the biological field, can solve the problems of simultaneous rupture of cell nucleus and mitochondria, and achieve the effect of high commercial application value, short time consumption and moderate lysis intensity

Pending Publication Date: 2021-06-01
SINGLERON NANJING BIOTECHNOLOGIES LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cell lysate provided by the present invention solves the problem of simultaneous rupture of the nucleus and mitochondria during the existing single cell lysis

Method used

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  • Single cell lysis buffer and application thereof
  • Single cell lysis buffer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Configuration of single cell lysate

[0021] The composition of the single-cell lysate, in specific applications, can use the ratio of the single-cell lysate components in the following example: Prepare 1mL single-cell lysate, and use a pipette to draw 50 uL of 2 mol / L Tris-Hcl into a centrifuge tube , add 5 mol / L Licl 100 uL, add 20% Triton X100 5uL, add 1 mol / L EDTA 20 uL, add 100mmol / LDTT50uL, blow and aspirate to mix, add sterile water 775 uL to make up to 1mL, mix and set aside.

Embodiment 2

[0022] Example 2: Configuration of single cell lysate

[0023] The composition of the single-cell lysate, in specific applications, can use the ratio of the single-cell lysate components in the following example: Prepare 1mL single-cell lysate, and use a pipette to draw 50 uL of 2 mol / L Tris-Hcl into a centrifuge tube , add 5 mol / L Licl 100 uL, add 20% Triton X100 5uL, add 1 mol / L EDTA 20 uL, add 100mmol / LDTT50uL, add sterile water 775 uL, mix and set aside.

Embodiment 3

[0024] Example 3: Lysis of 293T cells

[0025] The growth rate of 293T cells is fast, and the doubling time generally does not exceed 1 day. Subsequent experiments can be carried out after the recovery of the primary cells. Add 100 uL of the single-cell lysate prepared in Example 1 to the microfluidic chip, observe the cell lysis before and after addition under a microscope, record the lysis time, and take pictures to record the lysis process.

[0026] figure 1 is the microscope image of cells in the lysed microfluidic chip before lysis; figure 2 is the microscopic image of cells lysed in the lysed microfluidic chip; figure 1 , figure 2 It can be seen that before adding single-cell lysate, the outline of 293T cells falling into the microfluidic chip is clear, and the nucleus is clearly visible. After adding the single-cell lysate, the cells were completely lysed and there was almost no mitochondrial lysis, and the time for all the cells to be lysed was not more than 2 mi...

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PUM

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a single cell lysis buffer and application thereof. The single cell lysis solution provided by the invention can rapidly lyse cells while keeping mitochondria intact in a normal temperature state, is short in time consumption, and avoids RNA degradation and pollution caused by too high lysis temperature and long time consumption. Components of the single-cell lysis solution are common reagents, and the single-cell lysis solution is simple and convenient to prepare and low in cost. Through verification, the single cell lysis solution provided by the invention can realize single cell lysis only in 1-5 minutes at normal temperature, is efficient and simple, and has moderate lysis strength, and mitochondria is still intact after lysis; the single cell transcriptome sequencing success rate is obviously improved, and the method has high commercial application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a single-cell lysate and its application. Background technique [0002] Single-cell transcriptome sequencing (scRNA-seq) is a new technology that has emerged in recent years. It can obtain the expression profile of the whole transcriptome from the level of a single cell, perform high-throughput sequencing after amplification, and efficiently detect gene expression in a single cell. It has important application value in the development and differentiation of stem cells, diagnosis and treatment of tumors, and the design of targeted drugs. The single-cell sequencing process mainly includes single-cell extraction, cell lysis, mRNA reverse transcription, cDNA amplification, library construction, sequencing and data analysis. In recent years, microfluidic systems are mainly used to separate single cells from cell suspensions. After the single cells are obtained through microfl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6869
CPCC12Q1/6806C12Q1/6869C12Q2527/125C12Q2535/122
Inventor 訾晓渊雷佳
Owner SINGLERON NANJING BIOTECHNOLOGIES LTD
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