WO2020156049A1

WO2020156049A1 - Extraction liquid and application thereof in preserving tissue or cells and extracting rna

Info

Publication number: WO2020156049A1
Application number: PCT/CN2020/070563
Authority: WO
Inventors: 赵伟; 廖东升; 赵仲玖; 邱坤
Original assignee: 成都导胜生物技术有限公司
Priority date: 2019-02-01
Filing date: 2020-01-07
Publication date: 2020-08-06
Also published as: CN109593756A; CN109593756B

Abstract

Disclosed is an extraction liquid, which comprises an extraction liquid A and an extraction liquid B; the extraction liquid A is applied in the preservation of tissue or cells, and the extraction liquid A comprises 1.2-2.0 mol/L of guanidinium thiocyanate and 0.5-1.5 mol/L of guanidine hydrochloride; the extraction liquid B comprises 30-65vt% of water-saturated phenol and 9-15vt% of glycerin by volume fraction, the remainder being a solvent; the extraction liquid B further comprises 0.20-1 mol/L of guanidine thiocyanate, 0.4-0.8 mol/L of ammonium thiocyanate and 0.0073-0.018 mol/L of surfactant by molar concentration; and the pH of both the extraction liquid A and the extraction liquid B is 3.5-4.5. The extraction liquid B is applied in the extraction of RNA.

Description

An extracting solution and its application in preserving tissues or cells and extracting RNA

Technical field

The invention belongs to the technical field of biological preparations, and specifically relates to an extract and the application of the extract in preserving tissues or cells and extracting RNA.

Background technique

In modern molecular biology experiments and clinical molecular diagnosis, RNA is often isolated and purified from tissues and cells, and the quality of RNA often affects the success or failure of molecular biology experiments such as cDNA library, RT-PCR, and Northern blot.

Nowadays, the commonly used method is to first protect the tissue with tissue protection solution, and then use the extract solution to extract RNA from the tissue or cell. Commonly used protection methods include liquid nitrogen protection and protection solution protection, and the commonly used protection solutions include RNA later reagent and RNA safety reagent.

Among them, the liquid nitrogen protection method not only requires harsh conditions, but also has a high cost, which is only suitable for temporary tissue protection. The protective solution usually needs to be stored at low temperature, and the RNAlater solution is not compatible with the chemical components of TRIzol, silica gel column and magnetic bead method. The RNAlater solution contains ammonium isothiocyanate. When the tissue block is from the RNAlater solution When extracting the RNA for RNA extraction, if the RNAlater solution on the tissue block is not cleaned, the ammonium isothiocyanate in the RNAlater solution will bind or react with the phenol and SDS in the extract to form a precipitate, resulting in The composition and concentration of the extract change, and the lysis is not complete, which reduces the purity and yield of RNA. Therefore, tissues or cells preserved with RNA later solution usually need to be washed repeatedly before extraction, which wastes time and manpower and reduces extraction efficiency.

RNA extraction often uses TRIzol method, silica gel column, magnetic bead method, etc., but these methods generally suffer from low RNA yield and serious contamination of impurities, which cannot meet the quality of subsequent experiments such as genetic diagnosis, biochip analysis, and gene expression analysis. Claim.

In addition, the fragmentation process of the tissue mass also greatly affects the quality of RNA. Common methods for breaking up tissue blocks include liquid nitrogen grinding, homogenization (manual homogenization, machine homogenization, ultrasonic homogenization) and repeated freezing and thawing methods. The liquid nitrogen grinding method requires larger tissue blocks, and it is more difficult to completely break the tissue blocks; the homogenization method is more difficult to completely inhibit the endogenous RNase activity, which is easy to cause endogenous degradation; the disadvantage of repeated freezing and thawing is also difficult Large tissues are thoroughly lysed, and endogenous RNase activity is difficult to inhibit. Therefore, RNA extraction from animal-derived tissue blocks has difficulties such as complicated operations, incomplete fragmentation, and difficulty in inhibiting endogenous RNA activity, which makes it difficult to guarantee the quality and yield of RNA extraction.

Summary of the invention

In order to solve the problem of the incompatibility between the existing solution storage and the existing extraction solution in the prior art, which requires repeated cleaning, which results in low efficiency, the present invention provides an extraction solution and the extraction solution used in the preservation of tissues or cells and extraction Application in RNA.

The technical scheme adopted by the present invention is: an extracting solution comprising 1.2-2.0mol/L guanidine thiocyanate and 0.5-1.5mol/L guanidine hydrochloride, and the pH of the extracting solution is 3.5-4.5.

The application of the extract in the preservation of tissues or cells. First, the main components of the extract of the present invention are guanidine thiocyanate and guanidine hydrochloride. The guanidine thiocyanate is an organic compound with a molecular formula of C ₂ H ₆ N ₄ S, which is mainly used in biomedicine and chemical reagents.

At the same time, guanidine thiocyanate is a decoupling agent, a powerful protein denaturant, and destroys the secondary structure of proteins; it mainly plays a role in cell lysis and promotes the separation of nucleoprotein and nucleic acid; and acts as an enzyme inhibitor with a tissue preservation effect.

The guanidine hydrochloride is a white or slightly yellow mass, which is used as a strong denaturant for extracting total cellular RNA. Guanidine hydrochloride solution can dissolve protein, cause cell structure damage, nuclear protein secondary structure damage, and dissociate from nucleic acid. In addition, RNase can be inactivated by reducing agents such as guanidine hydrochloride. As a nuclease inhibitor, it can be Extract RNA from tissues containing RNase. In other words, guanidine thiocyanate can lyse tissues or cells, but at the same time, the increased guanidine hydrochloride can inhibit the activity of RNase to achieve the effect of lysis and preservation.

Through the quantitative configuration of guanidine thiocyanate and guanidine hydrochloride, it can provide tissues or cells with a certain period of room temperature preservation effect, and generally can be stored at room temperature for more than 7 days.

An extraction solution, including extraction solution A and extraction solution B;

The extract A includes 1.2-2.0 mol/L guanidine thiocyanate and 0.5-1.5 mol/L guanidine hydrochloride;

The extract B includes 30-65vt% water-saturated phenol and 9-15vt% glycerin in volume fraction, and the balance is solvent; extract B also includes 0.20-1mol/L thiocyanate in molar concentration Guanidine acid, 0.4-0.8mol/L ammonium thiocyanate and 0.0073-0.018mol/L surfactant;

The pH of the extraction liquid A and the extraction liquid B are both 3.5-4.5.

Further, the surfactant in the extract B is sodium lauryl sulfate.

Further, the pH of the extract A is adjusted with hydrochloric acid, and the pH of the extract B is adjusted with acetic acid.

Further, the extract B also includes β-mercaptoethanol with a molar concentration of 6.4 mmol/L to 19.20 mmol/L.

Further, the extract B also includes potassium citrate with a molar concentration of 0.05-0.15 mmol/L.

Further, the extract B also includes bromophenol blue with a molar concentration of 74.62 μmol/L-223.87 μmol/L.

Further, the extract B also includes sodium acetate with a molar concentration of 0.01-2 mol/L.

Further, the extract B also includes isopropanol with a volume fraction of 5-25%.

Further, the extract B also includes 8-hydroxyquinoline with a molar concentration of 0.689 mmol/L to 68.9 mmol/L.

Further, the extract B also includes DTT with a molar concentration of 0.5-1000 mmol/L.

Further, the extract B also includes urea with a molar concentration of 0.5-5 mol/L.

Further, the extract B also includes EDTA with a molar concentration of 0.1-500 mmol/L.

Further, the extract B also includes ammonium acetate with a molar concentration of 0.01-10 mmol/L.

The application of the extraction liquid in extracting RNA.

Sodium dihydrogen phosphate, sodium chloride and magnesium chloride can be used in the extraction solution as a buffer system to maintain the concentration of salt ions. The water-saturated phenol contained therein has the effect of lysing cells and precipitating proteins, and helps to remove DNA when it is acidic. Ammonium thiocyanate is a colorless crystal, easily deliquescent, soluble in water and ethanol, and soluble in methanol and acetone. Almost insoluble in chloroform and ethyl acetate, it can precipitate proteins, thereby achieving the effect of separating impurities; the surfactant uses SDS, namely sodium dodecyl sulfate, which has the effect of denaturing proteins; and by adjusting the pH value To 3.5-4.5 is mainly to ensure the quality of extraction, because too low pH will mainly reduce the purity of extraction, while too high will affect the yield.

The increased β-mercaptoethanol, which has the functional groups of ethylene glycol and ethanedithiol, is a volatile liquid with a strong pungent odor. βME is usually used for the reduction of disulfide bonds and can be used as an antioxidant in biological experiments. The reason why it is widely used is that its hydroxyl group enables it to be dissolved in water and reduces its volatility. In the present invention, it mainly serves to destroy the disulfide bond in RNase, thereby having an inhibitory effect.

Increased potassium citrate is a commonly used buffer. It is a white, slightly hygroscopic crystalline powder. Odorless, with the taste of saline, soluble in water, slowly soluble in glycerin, insoluble in alcohol, salty and cool in taste. It acts as a stabilizer and pH buffer, etc., participates in the buffer system, and acts as an enzyme inhibitor.

The molecular formula of the added bromophenol blue is C ₁₉ H ₁₀ Br ₄ O ₅ S, which is a light yellow to brownish yellow powder, and is usually used as an electrophoresis indicator dye, and is also used as a color indicator in the formulation of the present invention. During the centrifugation process, bromophenol blue sinks with DNA and protein, which can be used to indicate whether RNA isolation is complete.

The increased sodium acetate can precipitate sugars in the solution during the RNA extraction process, thereby achieving the effect of removing impurities and increasing the purity of the extracted RNA.

The added isopropanol is an organic compound, a colorless and transparent liquid, which protects the hydrophilic groups in the RNA chain through the hydrophobic effect of -OH, and has the effect of reducing the upper layer RNA.

Increased 8-hydroxyquinoline can also inhibit RNase, and the combined use with chloroform can enhance the inhibitory effect.

The increased DTT is a reducing agent, which is consistent with the effect of mercaptoethanol, which mainly destroys the disulfide bond in RNase, thereby having an inhibitory effect.

Increased urea is a white crystal-like organic compound; high-concentration urea can denature protein and can also inhibit RNase activity.

Increased EDTA is a divalent metal chelate and also acts as an enzyme inhibitor.

The structural formula of the added ammonium acetate is CH ₃ COONH ₄ , also known as ammonium acetate. It is a kind of white triangular crystal with acetic acid smell, which can be used as analytical reagent and meat preservative. In the present invention, the protein encountered in RNA extraction is precipitated, but the ammonium ion easily forms a white precipitate with the thiosulfate ion.

Among them, the extract A can store the tissue at room temperature for 24 hours to ensure that the RNA is not degraded, that is, it does not need to be stored at a low temperature and is free from the cold chain.

Further, the extraction method is:

G1. Tissues or cells preserved with extract A;

G2. Then add extract B to the tissues or cells protected by extract A, and mix upside down to obtain a mixture;

G3. When extracting RNA from tissues, ultrasonically disrupt the mixture obtained in step G2; when extracting RNA from cells, directly perform the operations in step G5 below;

G4. Mix the mixture after sonication upside down;

G5. Add chloroform to the mixture of RNA extracted from the cells in step G3 or the mixture of step G4, mix and centrifuge, and take the upper liquid;

G6. Add isopropanol to the upper liquid, mix well and centrifuge, discard the liquid to obtain a precipitate;

G7. Add ethanol solution to the precipitate, mix well and centrifuge, and discard the liquid;

G8. Add water to dissolve after the ethanol evaporates.

The water in the step G8 is double distilled water, deionized water or ultrapure water, the total mass of the extract A and the extract B is 100-150 times the mass of the tissue or cell, the ultrasonic power is 0.5-1000W, The ultrasound time is 0.5-30s. The volume of chloroform is 0.1-0.4 times the volume of the mixture. In the step G6, the volume ratio of isopropanol to the upper liquid is 1:1.

It is worth noting that all of the powdery components are calculated by molar concentration, so the remaining liquid components are calculated by volume fraction, and the powdery components are dissolved in the solvent, so the volume change is negligible.

The beneficial effects of the present invention are:

(1) The extract A of the present invention can replace the traditional RNA later solution for preserving tissues or cells. Therefore, when the extract A and the extract B are used to extract RNA, there is no need to wash the tissues or cells, which saves you The cleaning operation when storing RNA later solution simplifies the operation steps and significantly improves the extraction efficiency;

(2) The design of the distribution ratio of each group in the extract of the present invention is reasonable, and they have a synergistic effect with each other, so that the extracted RNA has high purity, high yield, and low protein pollution;

(3) The properties of the extract A and the extract B of the present invention are relatively stable, are not easily affected by conditions such as temperature and humidity, and can be fully used in conjunction to achieve the purpose of rapid, low-cost, high-quality, and large-scale extraction of animal RNA in the laboratory ；

(4) The newly added guanidine hydrochloride of the present invention has the effect of a nuclease inhibitor and can effectively extract RNA from tissues rich in RNase. The newly added sodium acetate has the effect of precipitating sugars encountered in RNA extraction, ensuring the purity of the extracted RNA. Newly added isopropanol: through the hydrophobic effect of -OH, the hydrophilic group in the RNA chain is protected, and at the same time has the effect of reducing the upper layer RNA;

(5) The present invention adjusts the amount of reagents for tissue preservation in extract A. The tissue can be stored at room temperature for 10 days or more. However, for liver tissues rich in RNase, the results of RNA extraction are normal, and the yield is improved. . In addition, it mainly increases multiple components in RNase pollution to prevent RNA from being degraded.

Description of the drawings

Figure 1 is a picture of agarose gel electrophoresis in Example 2 of the present invention.

detailed description

Example 1:

This embodiment provides an extraction solution, which includes 1.6 mol/L guanidine thiocyanate and 1 mol/L guanidine hydrochloride, and the pH of the extraction solution is adjusted to 4 by acetic acid. The extract is applied to preserve tissues or cells.

Example 2:

This embodiment provides an extraction liquid, including:

Among them, the extract is adjusted to pH 4 with acetic acid. The extract is used in RNA extraction.

Example 3:

This embodiment provides an extraction solution, including extraction solution A and extraction solution B;

Wherein the extract A includes 1.6 mol/L guanidine thiocyanate and 1 mol/L guanidine hydrochloride;

The extract B includes 57vt% water-saturated phenol, 0.24mol/L guanidine thiocyanate, 0.6mol/L ammonium thiocyanate, 11.25vt% glycerin and 0.01mol/L SDS; the remainder is solvent.

The pH of the extract A and the extract B are both 4.

The amount of RNA extracted using the extraction solution in this embodiment is 1000-1800 μg/mL, and it is measured that A260/A280 is in the range of 1.6-1.9, and A260/A230 is in the range of 1.6-1.9. The picture of agarose gel electrophoresis is shown in Figure 1.

The RNA extraction method using the above extraction solution A and extraction solution B, the specific steps are as follows:

1. Tissues or cells preserved in extract A, which contains 1.2-2.0 mol/L guanidine thiocyanate and 0.5-1.5 mol/L guanidine hydrochloride, and has a pH of 4.

2. Then add extract B to the tissues or cells stored in extract A, and mix upside down to obtain a mixture. The extract B includes 30-65vt% water-saturated phenol and 9-15vt% in volume fraction. Glycerin, the remainder is solvent; extract B also includes 0.20-1mol/L guanidine thiocyanate, 0.4-0.8mol/L ammonium thiocyanate and 0.0073-0.018mol/L surfactant in molar concentration , And the pH is adjusted to 4.

3. When extracting RNA from tissues, ultrasonically disrupt the mixture obtained in step 2; when extracting RNA from cells, proceed directly to the operation in step 5 below.

4. Mix the mixture after sonication upside down, where the ultrasonic power is 0.5-1000W, and the ultrasonic time is 0.5-30s.

5. Add chloroform to the mixture of RNA extracted from cells in step 3 or the mixture of step 4, mix and centrifuge, and take the upper liquid.

6. Add isopropanol to the upper liquid, mix well and centrifuge, discard the liquid to obtain a precipitate.

7. Add ethanol solution to the precipitate, mix well, centrifuge, and discard the liquid.

8. Add water to dissolve after the ethanol evaporates.

Experiment 1:

Take 8 1.5ml EP tubes, add 4mg rat liver tissue to each EP tube, and number each EP tube. Among them, add 250ul extract A to EP tubes 1, 3, 5 and 7, and store them at room temperature for 48h , Add 250ul RNAlater solution to No. 2, 4, 6, 8 EP tubes, and store them at room temperature for 48h.

Then add 750ul extract B to all the above EP tubes.

Invert the above-mentioned No. 1-8 EP tubes and perform ultrasonic ablation. The ultrasonic time is 5s, the frequency is 20kHz, and the power is 100W. Invert the EP tube after ultrasound several times to mix the mixture evenly; Add 200ul of chloroform to the EP tube, mix vigorously for 30 seconds, centrifuge at 10000g for 1 min, and take the upper liquid; pour the obtained upper liquid into another batch of clean EP tubes with corresponding numbers, and then add 500ul separately Isopropanol, slowly inverted and mixed for 50s, centrifuged at 10000g centrifugal force for 1 min, discarded the liquid, and obtained the precipitate; added 600ul of 75vt% ethanol solution to the above-mentioned precipitate, and slowly inverted and mixed for 30s, and then at 10000g centrifugal force Centrifuge for 2 minutes and discard the liquid; place the EP tube in the air to dry for 5 minutes and add 40ulddH2O to dissolve it; use a trace ultraviolet spectrophotometer to determine the OD value of the obtained RNA and agarose gel electrophoresis.

Obtain the data of experiment 1 and analyze the results. The results obtained can prove that the extraction solution A can obtain better quality RNA extraction purity than the existing RNAlater solution.

This application uses the A solution invented by our side, which increases and prolongs the tissue preservation time. The A solution invented by our side is used to compare the preservation effect with the RNA extraction reagent Trizol on the market. The mouse spleen tissue was stored at room temperature and 37°C. The storage time was set at more than 7 days. After the preservation, the tissue morphology was observed. It was found that the spleen tissue stored in Trizol had been completely dissolved. The solution A invented by our company to preserve the tissue Normally, RNA extraction was performed on the two kinds of preserved spleen tissues. As a result, the amount of RNA extracted by liquid A in this example was more than 3 times higher than that of Trizol preserved tissue.

This patent application was compared with the extraction results of the spleen tissue preserved by Trizol. The result was that the amount of RNA extracted from the tissue preserved by Trizol was 474μg/mL, and the measured A260/A280 was around 1.583 and A260/A230 was around 2.563. The amount of RNA extracted using the RNAlater of the present invention is 1241 μg/mL, and it is measured that A260/A280 is around 1.8 and A260/A230 is around 2.2.

Experiment 2:

Similarly, take 8 1.5ml EP tubes, add 4mg rat liver tissue to each EP tube, and number each EP tube. Among them, all EP tubes are added with 250ul RNAlater solution and stored at room temperature for 48h.

Then add 750ul extract B to the above-mentioned EP tubes 1, 3, 5, and 7 respectively; and add 1 ml of TRIzol solution to the EP tubes 2, 4, 6, and 8 respectively.

The experiment 1 data is obtained and the results are analyzed. The results obtained can prove that the extraction solution B can obtain higher quality RNA extraction purity than the existing TRIzol solution.

In this example, the RNA extracted after mixing extraction solution A and extraction solution B yields better quality RNA than the existing Trizol solution. The above two reagents are used to extract RNA from rat spleen tissue, and the extracted RNA For concentration determination, the amount of RNA extracted by Trizol is generally 400μg/mL, while the A260/A230 is determined, and the measured value is generally at the level of 1.5. The A260/A280 measurement result is at the 1.8 level. The amount of RNA extracted by the reagent of this embodiment is above 1200 μg/mL, while the A260/A230 is measured, and the measured value is generally at the 1.8 level. The A260/A280 measurement result is at the 1.7 level.

Experiment 3:

Add 750ul extract B to the above EP tubes 1, 3, 5, and 7 respectively; discard the RNA later solution in the above EP tubes 2, 4, 6, and 8, add 1×PBS to wash 3 times and discard the solution. Then add 1ml of TRIzol solution separately.

Using the mixed mode of preservation solution A and lysis solution B of this example, the extracted RNA has high extraction purity. The RNA extracted by our reagent is confirmed by 1% agarose gel electrophoresis. The RNA extracted by the reagent of this example The quality and effect are better than the RNA extracted by trizol. The result of the comparison between the two is that the amount of RNA extracted by Trizol is generally 400μg/mL, while the A260/A230 is measured, and the measured value is generally at the level of 1.5. The A260/A280 measurement result is at the 1.8 level. The amount of RNA extracted by the reagent of this embodiment is above 1200 μg/mL, while the A260/A230 is measured, and the measured value is generally at the 1.8 level. The A260/A280 measurement result is at the 1.7 level.

Experiment 4:

Take 6 1.5ml EP tubes, add about 106 Panc-1 cells to each EP tube, and number each EP tube. Among them, add 250ul extract A and 750ul extract B to EP tubes 1, 2, and 3 respectively. Add 1ml TRIzol to the 4, 5, and 6 EP tubes respectively; invert the above EP tubes several times to mix the mixture; add 200ul chloroform to the above EP tubes, respectively, vigorously invert and mix for 10 seconds, then centrifuge at 10000g 1min, take the upper liquid;

Pour the obtained upper layer liquid into another batch of clean EP tubes with corresponding numbers, and then add 500ul isopropanol, slowly mix upside down for 50s, centrifuge at 10000g centrifugal force for 1 min, discard the liquid, and obtain the precipitate; Add 600ul of 75vt% ethanol solution to the above-mentioned sediment, slowly mix by inversion for 30 seconds, centrifuge at 10000g centrifugal force for 1 min, discard the liquid; put the EP tube in the air to dry for 5 minutes, add 40ulddH2O to dissolve; the obtained RNA Micro-ultraviolet spectrophotometer to determine the OD value and agarose gel electrophoresis.

Using the reagent formulation of this example, RNA extraction and quality and purity evaluation were performed. The results of the spectrophotometer measurement and the results of agarose gel electrophoresis were compared. The amount of RNA extracted in this example was above 1200μg/mL. , At the same time, the A260/A230 are measured, and the measured value is generally at the 1.8 level. The A260/A280 measurement result is at the 1.7 level.

Example 4:

The extract B includes 57vt% water-saturated phenol, 0.24mol/L guanidine thiocyanate, 0.6mol/L ammonium thiocyanate, 11.25vt% glycerin, 0.3wt% SDS, 0.1wt% β- Mercaptoethanol, 0.08mmol/L potassium citrate, 0.1wt‰ bromophenol blue, 0.15mol/L sodium acetate, 8vt% isopropanol, 0.1wt% 8-hydroxyquinoline and 0.1mmol/L EDTA.

The pH of the extract A and the extract B were adjusted to 4 with acetic acid.

And for the above-mentioned best method, the experiment is carried out in a controlled variable manner by setting up multiple comparison groups.

Experimental group 1:

This embodiment provides an extraction solution, including extraction solution A and extraction solution B; wherein extraction solution A includes 1.6 mol/L guanidine thiocyanate and 1 mol/L guanidine hydrochloride;

The extract B includes 57vt% water-saturated phenol, 0.6mol/L ammonium thiocyanate, 11.25vt% glycerol, 0.3wt% SDS, 0.1wt% β-mercaptoethanol, 0.08mmol/L citric acid Potassium, 0.1wt% of bromophenol blue, 0.15mol/L of sodium acetate, 8vt% of isopropanol, 0.1wt% of 8-hydroxyquinoline, and 0.1mmol/L of EDTA.

The pH of the extract A and the extract B were adjusted to 4 with acetic acid.

Experimental group 2:

The extract B includes 57vt% water-saturated phenol, 0.24mol/L guanidine thiocyanate, 0.6mol/L ammonium thiocyanate, 11.25vt% glycerol, 0.1wt% β-mercaptoethanol, 0.08mmol/L L potassium citrate, 0.1 wt‰ bromophenol blue, 0.15 mol/L sodium acetate, 8 vt% isopropanol, 0.1 wt% 8-hydroxyquinoline and 0.1 mmol/L EDTA.

The pH of the extract A and the extract B were adjusted to 4 with acetic acid.

Experimental group 3:

The pH of the extraction liquid A and the extraction liquid B are both 7 or 2.

Experimental group 4:

The extract B includes 57vt% water-saturated phenol, 0.24mol/L guanidine thiocyanate, 0.6mol/L ammonium thiocyanate, 11.25vt% glycerin, 0.3wt% SDS, 0.08mmol/L lemon Potassium acid, 0.1wt% of bromophenol blue, 0.15mol/L of sodium acetate, 8vt% of isopropanol, 0.1wt% of 8-hydroxyquinoline and 0.1mmol/L of EDTA.

The pH of the extract A and the extract B were adjusted to 4 with acetic acid.

Experimental group 5:

The extract B includes 57vt% water-saturated phenol, 0.24mol/L guanidine thiocyanate, 0.6mol/L ammonium thiocyanate, 11.25vt% glycerin, 0.3wt% SDS, 0.1wt% β- Mercaptoethanol, 0.1wt‰ of bromophenol blue, 0.15mol/L of sodium acetate, 8vt% of isopropanol, 0.1wt% of 8-hydroxyquinoline, and 0.1mmol/L of EDTA.

The pH of the extract A and the extract B were adjusted to 4 with acetic acid.

Experimental group 6:

The extract B includes 57vt% water-saturated phenol, 0.24mol/L guanidine thiocyanate, 0.6mol/L ammonium thiocyanate, 11.25vt% glycerin, 0.3wt% SDS, 0.08mmol/L lemon Potassium acid, 0.1wt‰ of bromophenol blue, 0.15mol/L of sodium acetate, 8vt% of isopropanol, and 0.1mmol/L of EDTA.

The pH of the extract A and the extract B were adjusted to 4 with acetic acid.

Experimental group 7:

The extract B includes 57vt% water-saturated phenol, 0.24mol/L guanidine thiocyanate, 0.6mol/L ammonium thiocyanate, 11.25vt% glycerin, 0.3wt% SDS, 0.1wt% β- Mercaptoethanol, 0.08mmol/L potassium citrate, 0.1wt‰ bromophenol blue, 8vt% isopropanol, 0.1wt% 8-hydroxyquinoline, and 0.1mmol/L EDTA.

The pH of the extract A and the extract B were adjusted to 4 with acetic acid.

Experimental group 8:

The extract B includes 57vt% water-saturated phenol, 0.24mol/L guanidine thiocyanate, 0.6mol/L ammonium thiocyanate, 11.25vt% glycerin, 0.3wt% SDS, 0.1wt% β- Mercaptoethanol, 0.08mmol/L potassium citrate, 0.1wt‰ bromophenol blue, 0.15mol/L sodium acetate, 8vt% isopropanol, and 0.1wt% 8-hydroxyquinoline.

The pH of extract A and extract B was adjusted to 4 with acetic acid.

Experimental group 9:

The extract B includes 57vt% water-saturated phenol, 1mmol/L DTT, 0.24mol/L guanidine thiocyanate, 0.6mol/L ammonium thiocyanate, 11.25vt% glycerin, 0.3wt% SDS, 0.1wt% β-mercaptoethanol, 0.08mmol/L potassium citrate, 0.1wt‰ bromophenol blue, 5mol/L urea, 0.15mol/L sodium acetate, 8vt% isopropanol, 0.1wt% Of 8-hydroxyquinoline and 0.1mmol/L EDTA.

The pH of the extract A and the extract B were adjusted to 4 with acetic acid.

For the above-mentioned experimental groups lacking and adding different components, a unified experimental verification was carried out. Each group took 8 1.5ml EP tubes, and each EP tube was added with 4mg rat liver tissue, and each EP tube was numbered. Among them, all EP tubes Add 250ul of extract A to the tubes and store them at room temperature for 48h.

And add 750ul extract B to all the above EP tubes.

The results are analyzed as follows: The lack of guanidine thiocyanate in experimental group 1 directly caused insufficient lysis, which affected the constant of RNA extraction. The amount of RNA extracted is significantly reduced. Compared with the extraction results of the original formula, the amount of RNA extracted by the lack of 0.24mol/L guanidine thiocyanate reagent is less, and the amount of RNA extracted will be around 300μg/mL, and A260/A230 1.7 Up and down, A260/A280 is above 2, use a spectrophotometer for quantitative determination, and use agarose gel electrophoresis to determine the purity of the extracted RNA. In experimental group 2, the lack of 0.3wt% SDS directly led to insufficient lysis, which affected the constant of RNA extraction. The amount of RNA extracted is significantly reduced. Compared with the original extraction results, the amount of RNA extracted from the lack of 0.3wt% SDS reagent is too small. SDS directly affects the lysis. Quantitative determination is performed by spectrophotometer, and agarose gel electrophoresis is used for quantitative determination. The purity of the extracted RNA was judged, and the yield of RNA was around 200μg/mL.

In experimental group 3, the pH value changed, which not only affected the RNA yield, but also caused the extracted RNA to be impure and introduced genomic contamination. Using agarose gel electrophoresis to compare the purity of the extracted RNA, it can be found that the reagents that change the pH value have genome contamination. Changes in pH will directly cause genome contamination and will not affect RNA production. In experimental group 4, β-mercaptoethanol is used as a reducing agent to ensure that the ammonium sulfate in the welcome solution is not oxidized to ensure the stability of the reagent. The lack of 0.1wt% β-mercaptoethanol reagent will directly lead to unstable storage of the reagent and long storage time After that, the color of the reagent will change. The experimental group 5 lacked 0.08mmol/L potassium citrate, which would affect the reagent buffer system and thus affect the experimental results. The experimental group 6 lacks 8-hydroxyquinoline, because 8-hydroxyquinoline functions to maintain the stability of water-saturated phenols and prevent the oxidation of water-saturated phenols. Affect the long-term storage and stability of B liquid. In experimental group 7, the increase in sugar pollution affects the quality of RNA extraction. The lack of EDTA in experimental group 8 will increase the risk of RNA being degraded by RNase. Because 5mol/L urea and 1mmol/L DTT both inhibit the effect of RNase, so that the extracted RNA is protected from degradation. Therefore, the RNA stored in experimental group 9 has been degraded.

The present invention is not limited to the above-mentioned optional embodiments, and anyone can derive other products in various forms under the enlightenment of the present invention. The above-mentioned specific embodiments should not be construed as limiting the scope of protection of the present invention. The scope of protection of the present invention should be defined in the claims, and the description can be used to interpret the claims.

Claims

An extraction solution, characterized in that it comprises 1.2-2.0 mol/L guanidine thiocyanate and 0.5-1.5 mol/L guanidine hydrochloride, and the pH of the extraction solution is 3.5-4.5.
The use of an extract of claim 1 in the preservation of tissues or cells.
An extracting liquid, characterized in that it comprises extracting liquid A and extracting liquid B;

The extract A includes 1.2-2.0 mol/L guanidine thiocyanate and 0.5-1.5 mol/L guanidine hydrochloride;

The extract B includes 30-65vt% water-saturated phenol and 9-15vt% glycerin in volume fraction, and the balance is solvent; extract B also includes 0.20-1mol/L thiocyanate in molar concentration Guanidine acid, 0.4-0.8mol/L ammonium thiocyanate and 0.0073-0.018mol/L surfactant;

The pH of the extraction liquid A and the extraction liquid B are both 3.5-4.5.
An extraction solution according to claim 3, wherein the surfactant in the extraction solution B is sodium lauryl sulfate.
An extraction solution according to claim 3, wherein the extraction solution A uses hydrochloric acid to adjust the pH value, and the extraction solution B uses acetic acid to adjust the pH value.
The extract according to claim 3, wherein the extract B further comprises β-mercaptoethanol with a molar concentration of 6.4 mmol/L to 19.20 mmol/L.
The extract according to claim 3, wherein the extract B further comprises potassium citrate with a molar concentration of 0.05-0.15 mmol/L.
The extract according to claim 3, wherein the extract B further comprises bromophenol blue with a molar concentration of 74.62 μmol/L to 223.87 μmol/L.
An extraction solution according to claim 3, characterized in that: the extraction solution B further comprises sodium acetate with a molar concentration of 0.01-2 mol/L.
An extraction liquid according to claim 3, characterized in that: the extraction liquid B further comprises isopropanol with a volume fraction of 5-25%.
The extract according to claim 3, characterized in that: the extract B further comprises 8-hydroxyquinoline with a molar concentration of 0.689 mmol/L-68.9 mmol/L.
An extraction solution according to claim 3, characterized in that the extraction solution B further comprises DTT with a molar concentration of 0.5-1000 mmol/L.
An extraction solution according to claim 3, characterized in that: the extraction solution B further comprises urea with a molar concentration of 0.5-5 mol/L.
An extraction liquid according to claim 3, characterized in that: the extraction liquid B further comprises EDTA with a molar concentration of 0.1-500 mmol/L.
An extraction solution according to claim 3, characterized in that: the extraction solution B further comprises ammonium acetate with a molar concentration of 0.01-10 mmol/L.
The use of the extraction solution of any one of claims 3-15 in extracting RNA.