Cell storage liquid and preparation method of sputum cell reference product

A technology of preservation solution and reference product, which is applied in the field of nucleic acid preservation in vitro cells, can solve the problems of nucleic acid degradable formula and complex cost, and achieve the effect of protecting the integrity of nucleic acid, low cost and simple preparation method

Inactive Publication Date: 2018-06-01
ZHEJIANG JFK BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problem that the nucleic acids of cells and other tissue samples (such as sputum, etc.) in vitro are easy to degrade and the formula of the conventional cell preservation solution is com

Method used

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  • Cell storage liquid and preparation method of sputum cell reference product
  • Cell storage liquid and preparation method of sputum cell reference product
  • Cell storage liquid and preparation method of sputum cell reference product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Cells can be stored at room temperature (20°C) for 15 days using cell preservation solution

[0027] Step 1: Take 1ml of the A549 cell stock solution with a cell concentration of 2×106 / ml, centrifuge at 12000r / min for 1min, and discard the supernatant;

[0028] Step 2: Add 1ml of cell preservation solution, mix well, and place at room temperature (20°C);

[0029] Step 3: Observe the cell morphology under the microscope after 15 minutes, 5 days, 15 days and 20 days respectively, the results are shown in figure 1 . Depend on figure 1 It can be seen that the cells placed at room temperature (20°C) for 15 minutes, 5 days, and 15 days can still maintain a complete plump shape, and the cells after 20 days of storage are obviously lysed, and the plump shape of the cells disappears.

[0030] Therefore, the experimental results show that the cells can be preserved for 15 days at room temperature (20° C.) using the cell preservation solution.

Embodiment 2

[0031] Embodiment 2: Positive sputum reference product detection

[0032] Step 1: Collect the sputum of 20 patients with lung cancer confirmed by clinical pathology, add 10ml of sputum dissolving solution, shake at 37°C for 10 minutes to completely dissolve the sputum, centrifuge at 8000r / min for 5 minutes, and discard the supernatant;

[0033] Step 2: Resuspend the precipitate obtained in the previous step in 2ml of cell preservation solution, combine it into a 50ml centrifuge tube, mix well, transfer 50μl / tube to ten 1.5ml positive reference centrifuge tubes, numbered 1-10 respectively ;

[0034] Step 3: Using Telomerase Reverse Transcriptase Subunit (hTERT) mRNA Detection Kit" (National Machinery Note 20173404247), refer to the product manual to detect the sample obtained in step 2, after hybridization, washing, elution, Steps such as enzyme digestion and PCR amplification, the experimental results are shown in the table below.The results show that: the positive reference ...

Embodiment 3

[0037] Example 3: Cell precision reference product detection

[0038] Step 1: Take 1ml of cells to a concentration of 2×10 6 / ml of A549 cell stock solution, centrifuged at 12000r / min for 1min, discarded the supernatant, added 1ml of cell preservation solution to the pellet, and mixed thoroughly to obtain 2×10 6 / ml of A549 cell working solution;

[0039] Step 2: Take 50 μl of the working solution obtained in step 1, add 450 μl of cell preservation solution, and mix well to obtain 2×10 5 / ml A549 cell working solution, transfer 50μl / tube to ten 1.5ml high-concentration precision reference centrifuge tubes, numbered 1-10 respectively;

[0040] Step 3: Take 50 μl of the working solution obtained in step 2, add 450 μl of cell preservation solution, and mix well to obtain 2×10 4 / ml A549 cell working solution, transfer 50μl / tube to 10 1.5ml medium concentration precision reference centrifuge tubes, respectively numbered 1-10;

[0041] Step 4: Take 50 μl of the working solution ...

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Abstract

The invention provides a cell storage liquid having simple composition and aiming at maintaining nucleic acid stability in the cells, and a method for preparing a cell and sputum reference product byusing the cell storage liquid. The cell storage liquid is prepared from 75 percent ethanol, 0.9 percent of sodium chloride, 4 percent RNAsecure and the balance of diethyl pyrocarbonate treatment water. The method has the advantages that the cost is low; the preparation method is simple; no toxic and side effects exist. By using the method, the normal cell form can be maintained under the normal temperature condition (20 DEG C); meanwhile, the nucleic acid completeness in the cells can be effectively protected. The invention aims at providing a reference method for the preparation of a reference product of a nucleic acid detection type product of sputum or other tissue samples.

Description

technical field [0001] The method belongs to the technical field of nucleic acid preservation in in vitro cells, and relates to a simple and effective method for preparing a cell preservation solution and a sputum cell reference product aimed at preserving the stability of nucleic acid in cells. Background technique [0002] Cells are the basic structural and functional units of organisms. It is known that all organisms except viruses are composed of cells. Cells have high application value in biological research and medical fields. At present, the incidence of lung cancer and other cancers is increasing year by year, and nucleic acid or gene detection by shedding tumor cells has become a hot spot in medical diagnostic technology. Cells that leave the human body, or tumor cells that leave the culture environment, their physiological functions and cell morphology will change, and the nucleic acid in the cell is easily degraded, thus losing the significance of nucleic acid det...

Claims

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Application Information

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IPC IPC(8): A01N1/02G01N1/28
CPCA01N1/021G01N1/28
Inventor 陈燃王金固陈国强
Owner ZHEJIANG JFK BIOLOGICAL TECH
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