Biological sample whole RNA normal temperature long-term storage agent and preparation method and application thereof

Abstract

The application discloses a kind of biological sample whole RNA normal temperature long-term storage agent and its preparation method and application, belong to the technical field of biological medicine.The preservative includes the following components: tissue fixative, tissue penetrant, reactive biological sample encapsulant, siliconization blocking agent, pH regulator, ion concentration regulator and water.The preservative can be applied to bacteria, such as E. coli, Staphylococcus aureus;Cell, such as HEK293 cell, ID8 cell;Tissue, such as mouse liver tissue, human tumor tissue;Organ, such as small animal kidney, heart;Complete organism, such as earthworm, fruit fly and other different scale biological sample whole RNA normal temperature long-term storage, can guarantee RNA extraction efficiency and quality, especially suitable for field collection sample, clinical sample normal temperature transportation and long-term storage, can guarantee the extraction efficiency and quality of RNA in subsequent molecular experiment, has important significance to molecular biology research, clinical diagnosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a biological sample whole RNA normal temperature long-term preservation agent and its preparation method and application. BACKGROUND

[0002] Biological samples (such as viruses, bacteria, cells, organs and organisms, etc.) are the core experimental materials for molecular biology, genetics and clinical medicine research. As the key molecule for genetic information transmission in organisms, whole RNA is not only an important object for gene expression analysis (such as RT-PCR, RNA-seq) and protein synthesis regulation research, but also plays an irreplaceable role in disease diagnosis marker screening and pathogen detection.

[0003] However, RNA is more unstable than DNA and is easily degraded during biological sample collection, transportation and preservation: on the one hand, the RNase (RNAase) contained in the biological sample itself is difficult to inhibit after the sample is removed from the body and will quickly cut the RNA chain; on the other hand, external factors such as temperature fluctuations, oxidation, hydrolysis reaction and other external factors in the environment further exacerbate the degradation of RNA, leading to loss or distortion of genetic information, which seriously affects the reliability of subsequent experimental results. The current mainstream RNA preservation method for biological samples has significant defects, such as low-temperature freezing preservation which requires-20℃ / -80℃ refrigerator or liquid nitrogen environment, high equipment cost, high energy consumption, and sample devastating loss due to power failure or equipment failure, and professional cold chain equipment is required for transportation, which is difficult to meet the needs of outdoor collection, sample transportation in remote areas and other scenarios; traditional preservatives such as formalin and glutaraldehyde have low cost and good preservation effect, but they will cause RNA and protein cross-linking, hinder subsequent RNA extraction and interfere with reverse transcription and PCR reaction, which cannot meet the requirements of RNA quality for molecular experiments; existing normal temperature preservation technology partially realizes short-term normal temperature preservation by adding RNase inhibitors and dehydrating agents, but it has the problems of short preservation period (usually not more than 7 days) and limited sample size (only cells or small amount of tissue can be preserved).

[0004] Therefore, it is of great significance to develop a technology for normal temperature long-term preservation of biological sample whole RNA, wide sample size and high-quality and high-efficiency extraction of RNA, which can promote molecular biology research, clinical diagnosis and field scientific investigation. SUMMARY

[0005] The present application aims to overcome the problems of existing RNA preservation technology, such as dependence on low temperature, high cost, short preservation period, limited sample size, low extraction efficiency and quality, and provides a biological sample total RNA normal temperature long-term preservation agent. The preservation agent can realize the normal temperature long-term stable preservation of total RNA in biological samples of different sizes through the synergistic effect of multiple components, while ensuring the RNA extraction efficiency and quality. In addition, the preservation method and application of the preservation agent are also provided, which simplifies the operation steps, reduces the preservation cost, and meets the diversified scene demand.

[0006] Another object of the present application is to provide a preparation method of the above-mentioned biological sample total RNA normal temperature long-term preservation agent.

[0007] Still another object of the present application is to provide an application of the above-mentioned biological sample total RNA normal temperature long-term preservation agent.

[0008] The object of the present application is achieved by the following technical solutions:

[0009] A biological sample total RNA normal temperature long-term preservation agent, comprising the following components by mass fraction:

[0010] Tissue fixative: 10-1000 parts, preferably 100-1000 parts, more preferably 500-950 parts;

[0011] Tissue permeation agent: 10-1000 parts, preferably 10-400 parts, more preferably 10-200 parts;

[0012] Reaction type biological sample encapsulating agent (siliconization reagent): 1-150 parts, preferably 1-100 parts, more preferably 2-30 parts;

[0013] Siliconization blocking agent: 0.5-50 parts, preferably 1-30 parts, more preferably 2-15 parts;

[0014] pH regulator: 0.1-100 parts, preferably 0.1-50 parts, more preferably 1-20 parts;

[0015] Ion concentration regulator: 0.1-100 parts, preferably 0.1-50 parts, more preferably 1-20 parts;

[0016] Water: 10-500 parts, preferably 50-300 parts, more preferably 100-200 parts.

[0017] Further, the tissue fixative is selected from at least one of anhydrous methanol, anhydrous ethanol and acetone. It can quickly fix the morphology of biological samples, inhibit the endogenous RNase activity in the samples, and reduce the exposure and degradation of RNA caused by cell structure damage.

[0018] Furthermore, the tissue permeabilizer is selected from at least one of dimethyl sulfoxide (DMSO), glycerol, lauryl azone, isosorbide dimethyl ether, N-n-alkylbenzisothiazolone, and cyclohexanehexyl alcohol. It can penetrate into the sample, assisting the fixative and encapsulating agent in acting uniformly on the sample, while simultaneously enhancing the RNA's tolerance to environmental stress.

[0019] Furthermore, the reactive biological sample encapsulating agent (silicifying agent) is selected from silane compounds capable of forming inorganic / organic silicon nanolayers, specifically including at least one of methyltrimethoxysilane, methyltriethoxysilane, tetraethyl orthosilicate (TEOS), tetramethyl orthosilicate (TMOS), vinyltrimethoxysilane, and isobutyltriethoxysilane; preferably at least one of methyltriethoxysilane, tetraethyl orthosilicate, and tetramethyl orthosilicate, most preferably tetraethyl orthosilicate. It can form a dense silicon nanolayer on and inside the sample surface, isolating oxygen, moisture, and exogenous RNase, achieving long-term protection.

[0020] Furthermore, the silanization blocking agent is selected from at least one of hexamethyldisiloxane (HMDS), trimethylmethoxysilane, triethylmethoxysilane (TEMS), and tetraethylsilane (TES); preferably hexamethyldisiloxane. The silicon-oxygen bonds in its molecular structure can compete with the active groups (such as hydroxyl groups) of the silanizing reagent for binding, terminating the further silanization reaction and preventing the RNA from being encapsulated and difficult to extract due to an excessively thick silanized layer or excessive cross-linking. No additional reagents are needed for removal during subsequent extraction, and the RNA purity is not affected.

[0021] Furthermore, the pH adjuster is selected from at least one of hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid, sodium hydroxide, potassium hydroxide, ammonia, and Tris, and is used to adjust the pH of the preservative to 4.0–8.0 (preferably 5.5–7.0). This range can significantly inhibit RNase activity while avoiding damage to RNA structure from extreme pH.

[0022] Furthermore, the ion concentration regulator is selected from at least one of sodium chloride, potassium chloride, ammonium chloride, sodium phosphate, sodium monohydrogen phosphate, sodium dihydrogen phosphate, potassium phosphate, potassium monohydrogen phosphate, potassium dihydrogen phosphate, and sodium acetate. By adjusting the ion strength of the preservative (preferably 10-100 mmol / L), it maintains the native conformation of RNA and reduces RNA denaturation caused by changes in osmotic pressure.

[0023] Furthermore, the water is RNase-free pure water to avoid exogenous RNase contamination leading to RNA degradation.

[0024] The preparation method of the above-mentioned long-term room-temperature preservation agent for whole RNA of biological samples includes the following steps:

[0025] (1) Weigh the tissue fixative, tissue permeation agent, reactive biological sample encapsulant, siliconization blocking agent, pH adjuster, ion concentration adjuster and water by weight parts;

[0026] (2) Take 20% to 40% of the total amount of water, add the ion concentration adjuster, and stir until completely dissolved;

[0027] (3) Add the tissue fixative, tissue permeation agent, reactive biological sample encapsulant, and siliconization blocking agent in sequence, and stir until completely dissolved after adding each component;

[0028] (4) Add the remaining water, stir evenly, and make up the volume;

[0029] (5) Adjust the pH of the solution to 4.0 to 8.0 with the pH adjuster;

[0030] (6) Filter sterilization to obtain the biological sample whole RNA normal temperature long-term storage agent.

[0031] Further, step (5) is to adjust the pH of the solution to 5.5 to 7.0 with the pH adjuster.

[0032] The above-mentioned biological sample whole RNA normal temperature long-term storage agent is used in the application of biological sample whole RNA normal temperature long-term storage.

[0033] Further, the application includes the following steps: mixing the biological sample with the storage agent at a volume ratio of 1:1 to 1000, preferably 1:10 to 100, and more preferably 1:20 to 50, incubating at 0°C to 37°C for 5 minutes to 72 hours, preferably incubating at 25°C to 30°C for 1 hour to 48 hours, and then storing at normal temperature for a long time. After incubation, the stored sample can be directly placed in a normal temperature environment (15°C to 30°C) for long-term storage, or transported at normal temperature; when RNA needs to be extracted, the siliconization blocking agent does not need to be removed, and commercial RNA extraction kits (such as column-type animal tissue RNA extraction kits and bacterial RNA extraction kits) can be used directly to complete RNA extraction.

[0034] Further, the biological sample is any one of bacteria (such as Escherichia coli and Staphylococcus aureus), cells (such as HEK293 cells and ID8 cells), tissues (such as mouse liver tissue and human tumor tissue), organs (such as small animal kidneys and hearts), and whole organisms. This method is suitable for whole RNA normal temperature long-term storage of biological samples of different sizes, especially for normal temperature transportation and long-term storage of field-collected samples and clinical samples.

[0035] Further, the longest long-term storage time can be up to 60 days; preferably 45 to 60 days.

[0036] The present application has the following advantages and effects compared with the prior art:

[0037] The biological sample whole RNA normal-temperature long-term storage agent provided by the application can be applied to whole RNA normal-temperature long-term storage of biological samples of different scales such as bacteria (e.g., Escherichia coli and Staphylococcus aureus), cells (e.g., HEK293 cells and ID8 cells), tissues (e.g., mouse liver tissues and human tumor tissues), organs (e.g., small animal kidneys and hearts) and whole organisms (e.g., earthworms and fruit flies), can guarantee the RNA extraction efficiency and quality, is especially suitable for normal-temperature transportation and long-term storage of field-collected samples and clinical samples, can guarantee the RNA extraction efficiency and quality in subsequent molecular experiments, and has important significance for molecular biology research and clinical diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 is a scanning electron microscope (SEM) observation result diagram of cells; wherein, the left diagram is an untreated cell, and the right diagram is a cell after 3 h incubation at 25℃;

[0039] Figure 2 is a laser confocal microscope (Confocal) observation result diagram of cells;

[0040] Figure 3 is an observation diagram of the aggregation of untreated cells by an optical microscope;

[0041] Figure 4 is a storage condition diagram of cells stored at room temperature for 60 days;

[0042] Figure 5 is a half-life calculation result diagram of an aging experiment. DETAILED DESCRIPTION

[0043] In order to better understand the technical solutions of the application, the content of the application will be further described below in combination with examples, but the content of the application is not limited to the following examples only.

[0044] The performance test method of the application is as follows:

[0045] (I) Experimental materials

[0046] 1. Biological sample: ID8 mouse ovarian cancer cells

[0047] 2. Main reagents: anhydrous ethanol, dimethyl sulfoxide (DMSO), tetraethyl orthosilicate (TEOS), hexamethyldisiloxane (HMDS), Tris, sodium chloride, RNase-free water, UNIQ-10 column total RNA extraction kit (Shengwo), RNase inhibitor, agarose, SYBR Green qPCR Mix;

[0048] 3. Main instruments: scanning electron microscope (SEM), laser confocal microscope (Confocal), optical microscope, real-time fluorescence quantitative PCR instrument (qPCR instrument).

[0049] (II) Test method

[0050] 1. Sample morphology observation: SEM was used to observe the surface and internal structure of the sample after preservation to judge the formation of the silicified layer; laser confocal microscope (sample was labeled with DAPI for cell nucleus and FITC for silicified layer) was used to observe the uniformity and thickness of the silicified layer;

[0051] 2. Total RNA extraction and concentration determination: UNIQ-10 column total RNA extraction kit (product number: B511361) provided by Shenguo Biotechnology was used to extract RNA from the cell sample according to the standard protocol; the specific protocol is as follows: 1) take about 1×10 6 cells, centrifuge at 3000 rpm for 5 min, completely remove the supernatant, add 350 μL RLT Solution, mix thoroughly, homogenize with a homogenizer for 30 sec or aspirate 5 times with a 20-G (0.9 mm) needle; 2) add 1 / 2 volume of anhydrous ethanol to the lysed sample and mix thoroughly; 3) place the adsorption column in the collection tube, use a pipette to add the solution to the adsorption column, stand for 2 min, centrifuge at 8000 rpm for 1 min, discard the waste in the collection tube; 4) place the adsorption column back into the collection tube, add 500 μL RWSolution, stand for 1 min, centrifuge at 10,000 rpm for 1 min, discard the waste in the collection tube; 5) place the adsorption column back into the collection tube, add 500 μL RPE Solution, stand for 2 min, centrifuge at 10,000 rpm for 1 min, discard the waste in the collection tube; 6) repeat step 4) once; 7) place the adsorption column back into the collection tube, centrifuge at 10,000 rpm for 2 min; 8) place the adsorption column into an RNase-free 1.5 mL centrifuge tube, add 100 μL DEPC-treated ddH2O to the center of the adsorption membrane, stand for 5 min, centrifuge at 12,000 rpm for 2 min, and obtain the RNA solution; use the NanoDrop of Thermo Scientific to determine the total RNA concentration;

[0052] 3. RNA integrity detection: column animal tissue RNA extraction kit was used to extract RNA, 1% agarose gel electrophoresis was used to observe the integrity of the 28S rRNA and 18S rRNA bands of the RNA, and no obvious degradation bands were considered to be qualified;

[0053] 4. qPCR verification: reverse transcription of cDNA was performed using extracted RNA as a template, and qPCR reaction was performed using specific primers to detect the reverse transcription efficiency and subsequent amplification efficiency of RNA (CT value stable and less than 3 different from fresh sample is qualified);

[0054] ID8 mouse ovarian cancer cell RNA qPCR: template gene ACTB (GenBank: NM_007393.5), upstream primer: 5'-GGCTCCTAGCACCATGAAGA-3', downstream primer: 5'-AACGCAGCTCAGTAACAGTCC-3';

[0055] 5. Aging experiment determination: the siliconized ID8 mouse ovarian cancer cell samples or non-siliconized ID8 mouse ovarian cancer cell samples were transferred to a constant temperature and humidity box with 60% relative humidity and 3 different temperatures (55℃ / 60℃ / 65℃) for accelerated aging treatment; after different treatment times, DNA was extracted from the ID8 mouse ovarian cancer cell samples for analysis, and the half-life was calculated.

[0056] Example 1

[0057] Room temperature preservation of ID8 mouse ovarian cancer cell total RNA

[0058] (1) Preservative formula (by weight): 800 parts of anhydrous ethanol, 100 parts of DMSO, 7.5 parts of tetraethyl orthosilicate (TEOS), 5 parts of hexamethyldisiloxane (HMDS), 0.2 parts of Tris (adjusting pH to 6.5), 10 parts of sodium chloride, 70 parts of RNase-free water.

[0059] Preservative preparation:

[0060] 1) Weigh each component according to the above weight parts;

[0061] 2) Take 20 parts of RNase-free water (total water amount 70 parts, add 20 parts first), add 10 parts of sodium chloride, stir at 25℃ for 8 min until completely dissolved;

[0062] 3) Add 800 parts of anhydrous ethanol to the above solution, stir for 5 min until uniform, then add 100 parts of DMSO, continue to stir for 3 min;

[0063] 4) Add 7.5 parts of tetraethyl orthosilicate (TEOS), stir for 12 min to fully disperse the siliconizing reagent;

[0064] 5) Add 5 parts of hexamethyldisiloxane (HMDS) and stir for 6 min;

[0065] 6) Add the remaining 50 parts of RNase-free water, stir for 4 min, and then adjust the pH to 6.5 with 0.2 parts of Tris;

[0066] 7) Filter sterilization with 0.22 μm filter membrane.

[0067] (2) Preservation step: Take 1 × 10 6 ID8 mouse ovarian cancer cells, add 1 mL of the above-mentioned preservative, incubate at 25°C for 3 h, and then store in a normal temperature environment of 25 ± 2°C.

[0068] (3) Test results:

[0069] Morphology observation: SEM shows that a uniform thin siliconization layer is formed on the cell surface, without siliconization overexposure phenomenon Figure 1 ); laser confocal microscope shows that the siliconization layer is complete and uniform Figure 2 ); through optical microscope observation, it is shown that the untreated cells appear serious aggregation when placed at room temperature for a long time Figure 3 ).

[0070] RNA concentration and purity: The RNA concentration of the sample preserved for 0 days (fresh sample) is 135.2 ng / μL; the RNA concentration of the sample preserved for 1 day is 128.7 ng / μL; the RNA concentration of the sample preserved for 7 days is 112.3 ng / μL; and the RNA concentration of the sample preserved for 14 days is 86.3 ng / μL.

[0071] RNA integrity detection: Agarose gel electrophoresis shows Figure 4 ) that the 28 S and 18S rRNA bands of the RNA preserved for 60 days are still clearly visible, without obvious degradation;

[0072] qPCR verification: The CT value of the RNA preserved for 14 days after reverse transcription is 20.3, which is less than 1 different from that of the fresh sample (CT = 19.8), indicating that the reverse transcription and amplification efficiency of the RNA is stable.

[0073] Half-life estimation of aging experiment: The relative age of the cell total RNA preserved after siliconization is simulated through the accelerated aging experiment. It can be inferred that the RNA degradation half-life of the sample treated by the method is as long as 49 days, which is 652 times and 230 times of that of the unprotective group and the RNAlater (Thermo, AM7024) protective group, respectively Figure 5 ).

[0074] Comparative Example 1

[0075] Preservative without siliconization blocking agent

[0076] (1) Preparation of preservative: consistent with Example 1, but without hexamethyldisiloxane (HMDS);

[0077] (2) Preservation step: same as Example 1;

[0078] (3) Detection results:

[0079] Morphology observation: SEM showed that the silicon layer on the cell surface was too thick (thickness about 200-300 nm), and local cross-linking agglomeration appeared;

[0080] RNA concentration and purity: The RNA concentration was 133.6 ng / μL for 0 days (fresh sample), 73.7 ng / μL for 1 day, 67.3 ng / μL for 7 days, and 66.3 ng / μL for 14 days;

[0081] RNA integrity detection: Agarose gel electrophoresis showed that the 28 S and 18 S rRNA bands of the RNA stored for 60 days were still clear, and there was no obvious degradation;

[0082] qPCR verification: The CT value of the RNA stored for 14 days after reverse transcription was 25.6, which was significantly different from that of the fresh sample, indicating that excessive siliconization affected the extraction efficiency and quality of the RNA.

[0083] Comparative Example 2

[0084] Preservative without siliconization reagent

[0085] (1) Preparation of the preservative: same as Example 1, but without tetraethyl orthosilicate (TEOS);

[0086] (2) Preservation step: same as Example 1;

[0087] (3) Detection results:

[0088] RNA concentration and purity: The RNA concentration was 129.2 ng / μL for 0 days (fresh sample), 40.7 ng / μL for 1 day, 11.3 ng / μL for 7 days, and 6.3 ng / μL for 14 days;

[0089] RNA integrity detection: Agarose electrophoresis of the RNA stored for 7 days showed obvious degradation bands, and the 28 S rRNA band disappeared;

[0090] qPCR verification: The CT value of the RNA stored for 14 days after reverse transcription was 32.1, which could not meet the subsequent experimental requirements, indicating that the RNA was difficult to be stored for a long time without the protection of the silicon layer.

[0091] Comparative Example 3

[0092] Preservative without tissue fixative

[0093] (1) Preservative preparation: same as Example 1, but without ethanol;

[0094] (2) Preservation step: same as Example 1;

[0095] (3) Detection results:

[0096] RNA concentration and purity: the RNA concentration of the sample preserved for 0 day (fresh sample) was 134.7 ng / μL; the RNA concentration of the sample preserved for 1 day was 40.1 ng / μL; the RNA concentration of the sample preserved for 7 days was 22.1 ng / μL; and the RNA concentration of the sample preserved for 14 days was 11.8 ng / μL;

[0097] RNA integrity detection: the RNA agarose electrophoresis of the sample preserved for 7 days showed obvious degradation bands, and the 28 S rRNA band disappeared;

[0098] qPCR verification: the CT value of the sample preserved for 14 days after reverse transcription was 29.7, which could not meet the subsequent experimental requirements, indicating that the RNA was difficult to be preserved for a long time without the protection of the siliconized layer.

[0099] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods, and all shall be included in the protection scope of the present application.

Claims

1. A biological sample whole RNA room temperature long-term storage agent, characterized in that: consists of the following components by mass fraction: Anhydrous methanol: 800 parts; Dimethyl sulfoxide: 100 parts; Tetraethyl orthosilicate: 7.5 parts; Hexamethyldisiloxane: 5 parts; Tris: 0.2 parts; Sodium chloride: 10 parts; RNase-free pure water: 70 parts; The biological sample is an animal-derived cell, tissue or organ.

2. The method for preparing the biological sample total RNA long-term storage agent at room temperature as claimed in claim 1, characterized in that: It comprises the following steps: (1) Weigh anhydrous methanol, dimethyl sulfoxide, tetraethyl orthosilicate, hexamethyldisiloxane, Tris, sodium chloride and RNase-free pure water by mass fraction; (2) Take 20% to 40% of the total amount of RNase-free pure water, add sodium chloride, and stir until completely dissolved; (3) Add anhydrous methanol, dimethyl sulfoxide, tetraethyl orthosilicate and hexamethyldisiloxane in turn, and stir until completely dissolved after adding each component; (4) Add the remaining RNase-free pure water, stir evenly, and adjust the volume; (5) Adjust the pH of the solution to 6.5 with Tris; (6) Filter sterilization to obtain the biological sample whole RNA room temperature long-term storage agent; The biological sample is an animal-derived cell, tissue or organ.

3. The use of the biological sample total RNA room temperature long-term storage agent described in claim 1 in the room temperature long-term storage of biological sample total RNA, characterized in that: The biological sample is an animal-derived cell, tissue or organ.

4. Use according to claim 3, characterized in that: The application comprises the following steps: mixing the biological sample with the preservative at a volume ratio of 1:20 to 50, incubating at 25°C to 30°C for 1 hour to 48 hours, and then storing at room temperature for a long time.

5. The application of claim 3 or 4, characterized in that: The longest long-term storage time is 60 days.

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