Treatment method for room-temperature preservation of cell deoxyribonucleic acid

Abstract

A treatment method for room-temperature preservation of cell deoxyribonucleic acid, comprising the following steps: placing cells in a preservation tube containing a cell deoxyribonucleic acid preservation reagent, and performing freezing preservation at a temperature of -20°C to -80°C; and then performing thawing, and drying the cell deoxyribonucleic acid preservation reagent within the preservation tube to eliminate moisture.

Description

Methods for processing deoxyribonucleic acid (DNA) of cells for room temperature storage Technical Field

[0001] This invention relates to a method for processing cellular deoxyribonucleic acid (DNA) for room temperature preservation. Background Technology

[0002] In existing technologies, when transporting cellular deoxyribonucleic acid (DNA) to experimental or testing units, due to the low amount of DNA, samples are transported frozen to prevent loss and maintain DNA integrity, thus fixing the samples at the bottom of the preservation tube. Current commercially available pharmaceutical cold chain transportation requires monitoring temperature changes during transport, and manual adjustments or replenishment are needed when temperatures are too high or cooling materials (such as dry ice or coolant bags) are depleted. Therefore, the cost per shipment, whether for short-distance domestic or long-distance international transport, is significantly higher than room temperature transport, especially when only one sample is being transported at a time. In view of this, the purpose of this invention is to provide a method for long-distance, room-temperature transport of cellular DNA.

[0003] Summary of the Invention

[0004] This invention provides a method for preserving cellular deoxyribonucleic acid (DNA), which enables long-distance, long-term transport of cellular DNA at room temperature.

[0005] The present invention provides a method for preserving cellular deoxyribonucleic acid (DNA), comprising the following steps: Cells are placed in a preservation tube containing a DNA preservation reagent and frozen at a temperature of -20°C to -80°C. Subsequently, the cells are thawed, and the DNA preservation reagent in the preservation tube is subjected to a drying process to remove moisture.

[0006] In one embodiment of the present invention, the freezing time at a temperature of -20°C to -80°C is more than 3 hours.

[0007] In one embodiment of the present invention, 1 to 100,000 cells are placed in a preservation tube containing 1 to 20 microliters of cell deoxyribonucleic acid preservation reagent.

[0008] In one embodiment of the present invention, the cell deoxyribonucleic acid preservation reagent is an isotropic solution of the cells.

[0009] In one embodiment of the present invention, the cell deoxyribonucleic acid preservation reagent includes sodium chloride, acetyl-L-carnitine, α-lipoic acid or N-acetyl-L-cysteine, proclin 300, glutathione, vitamin E or Tris-HCl.

[0010] In one embodiment of the present invention, after removing moisture, the cell deoxyribonucleic acid is preserved for at least 11 days at a temperature of 15°C to 35°C.

[0011] In one embodiment of the invention, the drying process includes passing through a water bath, a heating plate, placing the device in a ventilated biosafety cabinet to increase airflow, or a centrifugal concentrator.

[0012] In one embodiment of the invention, the temperature is raised to 35°C to 95°C by means of a water bath or a heating plate.

[0013] In one embodiment of the invention, a centrifugal concentrator is used to perform a drying process at a temperature of 15°C to 55°C.

[0014] Based on the above, this invention provides a method for preserving cellular deoxyribonucleic acid (DNA), which can preserve cellular DNA for extended periods at room temperature. Therefore, compared to existing pharmaceutical cold chain transportation methods, this invention's method for preserving cellular DNA can effectively reduce transportation costs and maintain the integrity of cellular DNA. Detailed Implementation

[0015] The embodiments of the present invention will now be described in detail. However, these embodiments are illustrative and the present invention is not limited thereto.

[0016] In this document, the range expressed as "from one value to another" is a concise way of representing a range to avoid listing all the values ​​in that range in the specification. Therefore, the description of a particular range of values ​​encompasses any value within that range as well as the smaller range of values ​​defined by that value, just as the arbitrary value and the smaller range of values ​​would be written in the explanatory text of the specification.

[0017] This invention provides a method for preserving cellular deoxyribonucleic acid (DNA), comprising the following steps: Cells are placed in a preservation tube containing a DNA preservation reagent and frozen at a temperature of -20°C to -80°C. Subsequently, the cells are thawed, and the DNA preservation reagent in the preservation tube is subjected to a drying process to remove moisture.

[0018] In this embodiment, cells are frozen at a temperature of -20°C to -80°C for, for example, more than 3 hours. For example, 1 to 100,000 cells are placed in a preservation tube containing, for example, 1 to 20 μL of cell deoxyribonucleic acid (DNA) preservation reagent. However, this invention is not limited to this, and the amount of cells and DNA preservation reagent added can be adjusted according to actual operating conditions. The preservation tube is, for example, a 0.2 mL PCR tube. The DNA preservation reagent is an isotropic solution of the cells and may include sodium chloride, acetyl-L-carnitine, α-lipoic acid or N-acetyl-L-cysteine, proclin 300, glutathione, vitamin E, or Tris-HCl to protect cell morphology, the integrity and stability of intracellular DNA. However, this invention is not limited to this.

[0019] In this embodiment, after moisture removal, the cellular deoxyribonucleic acid (DNA) is preserved for at least 11 days at a temperature of 15°C to 35°C, maintaining its integrity for subsequent experiments or assays. The drying process may include using a water bath, heating plate, placing the sample in a ventilated biosafety cabinet to increase airflow, or a centrifuge to raise the temperature to, for example, 35°C to 95°C, or using a centrifuge to perform the drying process at a temperature of 15°C to 55°C. After moisture removal, visible crystals of the components in the DNA preservation reagent can be observed and are visible at the bottom of the preservation tube.

[0020] Table 1 below lists the drying conditions for a 3 μL sample:

[0021] Table 1

[0022] The following experimental examples illustrate in detail the method for preserving cellular deoxyribonucleic acid (DNA) proposed in the above embodiments. Samples transported at room temperature for 11 days using this method still retain good cellular DNA integrity and can be used for subsequent experimental detection, such as preimplantation genetic testing (PGT) and other DNA-related detection experiments. However, the following experimental examples are not intended to limit the invention.

[0023] Sample preparation

[0024] This test included a total of 7 blastocysts. For each blastocyst, 3 to 10 cells were collected twice as test samples and divided into two groups: Day 1 (control group) and Day 11 (test group). Cells were stored in 3 μL of 1X phosphate-buffered saline without calcium and magnesium. After sample preparation, both groups were immediately stored at -80°C. After 24 hours, both groups were removed and briefly centrifuged. The samples were then dried using a centrifuge. The Day 1 group samples were immediately returned to the -80°C freezer, while the Day 11 group samples were left at room temperature for 11 days. After 11 days, both groups underwent preimplantation chromosome number screening.

[0025] Preimplantation chromosome number screening (PGT-A)

[0026] PGT-A assays were performed according to the Pre-Implantation Methylation Screening (PIMS) manual guidelines (Guangzhou Nuwa Life Science Technology Co., Ltd.). In short, genomic DNA was extracted from 3 to 10 cells of the blastocyst and fragmented via enzymatic digestion. The fragmented DNA was then subjected to end repair, dA tailing, and adapter ligation. After 15 cycles of PCR amplification using indexed sequencing primers, the PCR-amplified samples were sequenced using an Illumina NextSeq 550 system. The copy number of each chromosome in each sample was calculated using PIMS analysis software. The experimental results are shown in Table 2 below.

[0027] Table 2

[0028] In summary, this invention provides a method for preserving cellular deoxyribonucleic acid (DNA). By placing cells in preservation tubes containing a DNA preservation reagent and freezing them at -20°C to -80°C, followed by thawing and drying to remove moisture, cellular DNA can be preserved at room temperature for an extended period. Specifically, at temperatures between 15°C and 35°C, cellular DNA can be preserved for at least 11 days. This effectively maintains the integrity of the cellular DNA for subsequent experiments or testing. Therefore, compared to existing pharmaceutical cold chain transportation methods, this method for preserving cellular DNA can effectively reduce transportation costs, especially international transportation costs.

Claims

1. A method for preserving cellular deoxyribonucleic acid, characterized in that, comprising: freezing the cells in a storage tube containing a cell deoxyribonucleic acid storage reagent at a temperature of -20°C to -80°C; and thawing and performing a drying procedure on the cell deoxyribonucleic acid storage reagent in the storage tube to remove water.

2. The method of claim 1, wherein the cell DNA is preserved. The freezing of the cells at a temperature of -20°C to -80°C is for a period of 3 hours or more.

3. The method of claim 1, wherein the cell DNA is preserved. 1 to 100,000 of the cells are placed in the storage tube containing 1 microliter to 20 microliters of the cell deoxyribonucleic acid storage reagent.

4. The method of claim 1, wherein the cell DNA is preserved. The cell deoxyribonucleic acid storage reagent is an isotonic solution for the cells.

5. The method of claim 4, wherein the cell DNA is preserved. The cell deoxyribonucleic acid storage reagent comprises sodium chloride, acetyl-L-carnitine, alpha-lipoic acid or N-acetyl-L-cysteine, proclin 300, glutathione, vitamin E or Tris-HCl.

6. The method of claim 1, wherein the cell DNA is preserved. After the removal of water, the cell deoxyribonucleic acid is stored for at least 11 days at a temperature of 15°C to 35°C.

7. The method of claim 1, wherein the cell DNA is preserved. The drying procedure comprises a water bath, a heating plate, placement in a fumehood biosafety cabinet to increase air flow or a centrifugal concentrator.

8. The method of claim 7, wherein the cell DNA is preserved. The temperature is increased to 35°C to 95°C by the water bath or the heating plate.

9. The method of claim 7, wherein the cell DNA is preserved. The drying procedure is performed using the centrifugal concentrator at a temperature of 15°C to 55°C.

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