阅读说明：本技术 一种核酸保护试剂及其使用方法 (Nucleic acid protection reagent and use method thereof ) 是由 不公告发明人 于 2019-09-18 设计创作，主要内容包括：本发明提供了一种核酸保护试剂,其包括含戊二醛、乙醇的DEPC水溶液、含EDTA、甘氨酸、海藻糖、Tris碱的DEPC水溶液。本发明还提供了使用该试剂保存生物样本中包括cfDNA和exRNA在内的DNA和RNA的方法。(The invention provides a nucleic acid protection reagent which comprises a DEPC aqueous solution containing glutaraldehyde and ethanol, and a DEPC aqueous solution containing EDTA, glycine, trehalose and Tris alkali. The present invention also provides a method for preserving DNA and RNA including cfDNA and exRNA in a biological sample using the reagent.)

1. A nucleic acid protecting agent comprising the following components:

A. a solution containing glutaraldehyde and ethanol prepared by DEPC water;

B. a solution containing EDTA, glycine, trehalose, Tris base, formulated with DEPC water.

2. The nucleic acid protection kit according to claim 1, wherein:

A. a solution containing 5-15% v/v glutaraldehyde and 30-50% v/v ethanol prepared with DEPC water;

B. a solution prepared using DEPC water and containing EDTA 5-15% w/v, glycine 3-9% w/v, trehalose 4-12% w/v, Tris base 0.5-5% w/v.

3. The nucleic acid protection kit according to claim 2, wherein:

A. a solution containing glutaraldehyde 10% v/v, ethanol 40% v/v, formulated with DEPC water;

B. a solution containing EDTA 10% w/v, glycine 5% w/v, trehalose 7% w/v, Tris base 1% w/v, formulated with DEPC water.

4. A method for preserving a biological sample using the nucleic acid protection kit according to any one of claims 1 to 3, which comprises treating the sample with solution A, adding solution B, and preserving for a long period of time.

5. The method according to claim 4, wherein the biological sample is a tissue, cell, blood, body fluid, soil or feces.

6. The method according to claim 5, wherein when the biological sample is a solid, the solution A is added in an amount sufficient to immerse the biological sample, and after 20 minutes of fixation, the solution B is added in an amount 3 times the amount of the solution A and stored.

7. The method according to claim 5, wherein when the biological sample is a liquid, the amount of the solution A is 0.5 to 2 times the volume of the biological sample, and the solution B is 3 times the amount of the solution A after the fixation for 10 minutes.

8. A method according to any one of claims 4 to 7 wherein the stored objects comprise DNA, RNA.

9. The method according to claim 8, wherein the preserved objects further comprise cfDNA, exRNA.

Technical Field

The invention belongs to the field of molecular biology, and particularly provides a nucleic acid protection kit which comprises a DEPC aqueous solution containing glutaraldehyde and ethanol, and a DEPC aqueous solution containing EDTA, glycine, trehalose and Tris alkali. The invention also provides a method for preserving DNA and RNA including cfDNA and exRNA in a biological sample by using the kit.

Background

With the development of molecular biology, nucleic acid has become an increasingly important target molecule in various scientific researches and diagnostic tests. Nucleic acid, particularly small nucleic acid molecules such as cfDNA and exRNA are easily inactivated and distorted by external conditions or nuclease during processing and storage, so that the storage of nucleic acid in a biological sample is an important guarantee for molecular biological research and detection.

There are many reagents/kits for preserving nucleic acids on the market, such as RNA laters, RNA tables, etc., but some of these reagents require cryopreservation, some require extraction steps (difficult to achieve in areas with large sample size and poor handling conditions), some are dedicated to certain biological samples, and are expensive to sell. Therefore, the search for a nucleic acid protective reagent which can stably preserve a biological sample at normal temperature for a long time without using special reagents and heavy instruments and maintain the nucleic acid status therein is of great significance for the promotion of large-scale disease screening/the implementation of related scientific research.

Disclosure of Invention

The applicant finds that when a cell sample or a blood sample is treated, a small amount of cross-linking agent/protein denaturant such as glutaraldehyde and ethanol is added to partially cross-link/primarily fix proteins on the surface of the sample, and then a preservation solution containing a chelating agent, a protective agent and the like is added, so that a biological sample can be preserved for a long time at normal temperature while DNA and RNA including cfDNA and exRNA are basically unchanged.

In one aspect, the present application provides a nucleic acid protection kit comprising the following components:

A. a solution containing glutaraldehyde and ethanol prepared by DEPC water;

B. a solution containing EDTA, glycine, trehalose, Tris base, formulated with DEPC water.

Further, wherein:

A. a solution containing 5-15% v/v glutaraldehyde and 30-50% v/v ethanol prepared with DEPC water;

B. a solution prepared using DEPC water and containing EDTA 5-15% w/v, glycine 3-9% w/v, trehalose 4-12% w/v, Tris base 0.5-5% w/v.

Further, wherein:

A. a solution containing glutaraldehyde 10% v/v, ethanol 40% v/v, formulated with DEPC water;

B. a solution containing EDTA 10% w/v, glycine 5% w/v, trehalose 7% w/v, Tris base 1% w/v, formulated with DEPC water.

In another aspect, the present application provides a method for preserving a biological sample using the nucleic acid protection kit, comprising treating the sample with solution A, and adding solution B for long-term preservation.

Further, the biological sample is a tissue, a cell, blood, a body fluid, soil or feces.

Further, when the biological sample is a solid, a solution A enough to immerse the biological sample is added, and after 20 minutes of fixation, a solution B3 times the amount of the solution A is added for preservation. .

Further, when the biological sample is liquid, liquid A with the volume 0.5-2 times of the volume of the biological sample is added according to different samples, and liquid B with the volume 3 times of the liquid A is added after fixing for 10 minutes for preservation.

Further, the stored objects comprise DNA and RNA.

Further, the preserved objects also comprise cfDNA and exRNA.

The nucleic acid protection reagent group which is simple to operate, does not need any instrument, is low in price, does not use special inhibitor and enzyme, has low storage requirement, can be stored for a long time at 4 ℃ and even at normal temperature, and has important significance for large-scale disease screening/detection, particularly disease screening and detection under poor operating conditions.

Drawings

The figure shows the electrophoresis of RNA extracted from HCT 116 after 0, 7, 14 and 21 days of storage.

Detailed description of the preferred embodiments