Unlock instant, AI-driven research and patent intelligence for your innovation.

FFPE sample pretreatment liquid and method for separating single cells from FFPE sample

A technology for pretreatment liquid and sample, applied in cell dissociation methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of many operation steps and time-consuming, and achieve a simple process, short time-consuming, and strong practicability. Effect

Pending Publication Date: 2021-06-11
SINGLERON NANJING BIOTECHNOLOGIES LTD
View PDF0 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are methods for separating FFPE samples into single cells, but all of them are time-consuming, have many operation steps, and need to be sorted by flow cytometry. There is an urgent need for a fast and simple method for separating single cells in FFPE tissues. single cell RNA sequencing

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • FFPE sample pretreatment liquid and method for separating single cells from FFPE sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: configuration of pretreatment liquid

[0027] The composition of the FFPE sample pretreatment solution can be proportioned according to the following examples in specific applications: The FFPE sample pretreatment solution contains 50 units / mL collagenase type iv (Collagenase Type 4), 80 units / mL collagenase type II (collagenase Type 2), 0.6 units / mL dispase I (Dispase I), 100 units / mL hyaluronidase (hyaluronidase), 0.2 units / µL marine RNase inhibitor, 0.5 mM calcium chloride (CaCl 2 ) and 5 mM dithiothreitol (DTT) in PBS buffer.

Embodiment 2

[0028] Embodiment 2: configuration of pretreatment liquid

[0029] The components of the FFPE sample pretreatment solution can be proportioned according to the following examples in specific applications: The FFPE sample pretreatment solution contains 35 units / mL Collagenase Type 4, 60 units / mL collagenase Type 2, and 0.4 units / mL Dispase I , 70 units / mL hyaluronidase, 0.1 units / µL marine RNase inhibitor, 0.3mM CaCl 2 and 4mM dithiothreitol (DTT) in PBS buffer.

Embodiment 3

[0031] In this embodiment, mouse liver FFPE tissue is used as a test sample, and a method for isolating single cells from formalin-fixed paraffin-embedded tissue is provided, and the specific steps are as follows:

[0032] (1) Slicing: Use a scalpel to remove excess paraffin from both sides of the mouse liver FFPE tissue, and cut the FFPE tissue into thin slices of about 40 µm;

[0033] (2) Dewaxing: Collect the slices into a 1.5 mL centrifuge tube, soak and wash them with 1 mL xylene for 3 times (the first time the slices need to be soaked in 55°C for 3 minutes to accelerate the dissolution of paraffin, and the last two times at room temperature Soak for 5min) to remove residual paraffin;

[0034] (3) Rehydration: immerse the slice in absolute ethanol × 2, 95% ethanol, 70% ethanol, 50% ethanol, 30% ethanol in turn, 5 minutes each time;

[0035] (4) Wash twice in 1 mL Tris-EDTA (1×TE, pH 8.0) solution, treat in a water bath at 70°C for 20 minutes to reverse protein-nucleic ac...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
thicknessaaaaaaaaaa
Login to View More

Abstract

The invention relates to the field of single cell omics analysis, in particular to an FFPE sample pretreatment liquid and a method for separating single cells from an FFPE sample. The FFPE sample pretreatment liquid provided by the invention is a common molecular cell biological reagent, and the RNA enzyme inhibitor is added to effectively inhibit the degradation of RNA in a sample; the method for separating single cells from a formalin-immobilized paraffin embedded sample is simple in process, short in time consumption and low in reagent material consumption; a flow cytometer does not need to be used for sorting, the requirement for equipment is low, single cells in the formalin fixed paraffin embedded sample can be separated in a high-quality manner in a common laboratory, and the separation cost is effectively reduced; and the whole separation process only needs 3 to 5 hours, so that the method has very strong practicability and can obviously improve the accuracy of single-cell RNA (Ribonucleic Acid) sequencing.

Description

technical field [0001] The invention relates to the field of single-cell omics analysis, in particular to a FFPE sample pretreatment solution and a method for separating single cells from the FFPE sample. Background technique [0002] Single-cell RNA sequencing (scRNA-seq) can reveal the uniqueness of each cell, thereby studying biology at the single-cell molecular level and solving previously unanswered questions, such as revealing cell heterogeneity, cell development, different genes between cells Expression patterns, etc., have been widely used in various fields such as biology, pharmaceutical research and development, and clinical medicine. The scRNA-seq method includes the steps of (1) isolation and lysis of single cells or nuclei, (2) reverse transcription, (3) cDNA amplification, and (4) preparation of sequencing libraries. Currently, isolation and lysis of single cells or nuclei requires detachment from the extracellular matrix and cell-cell adhesions (except for ci...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/00
CPCC12N5/00C12N2509/00
Inventor 訾晓渊丁修恒尤园园朱文奇
Owner SINGLERON NANJING BIOTECHNOLOGIES LTD