CfRNA precipitator, body fluid cfRNA and body fluid cfRNA extraction method

Abstract

The invention provides a cfRNA precipitator, body fluid cfRNA and a body fluid cfRNA extraction method, and belongs to the technical field of biology. According to the method, the absolute ethyl alcohol, the ammonium acetate and the linear acrylamide are used for preparing the cfRNA precipitant, the prepared cfRNA precipitant has high affinity to cfRNA and low adsorption to protein and DNA, the cfRNA precipitant is used for extracting cfRNA in body fluid, and the obtained cfRNA is high in purity and good in quality. The body fluid cfRNA extraction method provided by the invention can adapt to various complex biological body fluid samples, provides powerful technical support for liquid biopsy and disease diagnosis, can improve the yield and purity of the cfRNA, reduce the sample treatment time, enhance the operation convenience, and ensure the molecular integrity of the cfRNA so as to facilitate accurate downstream analysis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a cfRNA precipitant, body fluid cfRNA and a method for extracting body fluid cfRNA. Background Art

[0002] Extracellular RNA (cfRNA) is a free-living nucleic acid that exists outside cells in human body fluids. It is closely related to cell metabolism and can reflect the physiological and pathological conditions of cells. With the development of precision medicine, liquid biopsy technology based on cfRNA is gradually becoming a research hotspot due to its advantages such as non-invasiveness and repeatability. The detection of cfRNA levels is of great significance in prenatal diagnosis, early diagnosis of malignant tumors, and disease course monitoring. By analyzing cfRNA in body fluids, key information can be provided for early diagnosis of diseases, treatment monitoring, and efficacy evaluation.

[0003] However, due to the diversity and complexity of body fluid sample types, traditional cfRNA extraction methods face challenges such as low efficiency, low purity, and limitation to specific sample types. Therefore, how to efficiently extract high-quality cfRNA from different types of body fluid samples has become an urgent problem to be solved. Summary of the invention

[0004] The object of the present invention is to provide a cfRNA precipitant, body fluid cfRNA and a method for extracting body fluid cfRNA. The cfRNA extracted using the cfRNA precipitant of the present invention has high purity and good quality.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The invention provides a cfRNA precipitant, which consists of anhydrous ethanol, ammonium acetate and linear acrylamide in a volume ratio of 225-400:10-20:1.

[0006] The present invention also provides a method for extracting cfRNA from body fluid using a cfRNA precipitant, comprising the following steps: (1) After pretreatment of the body fluid, centrifuge and continue to add RNA carrier to mix evenly, then add lysis buffer and digestion buffer, mix evenly, and heat in a water bath to obtain a mixed solution; (2) adding a precipitant to the mixed solution for precipitation, using an adsorption column for centrifugal adsorption, washing the adsorption column with a washing solution, and eluting with an eluent to obtain a cfRNA solution.

[0007] Furthermore, in step (1): When the body fluid is serum or plasma, the pretreatment method is: subjecting blood to plasma treatment or serum treatment to obtain serum or plasma; When the body fluid is cerebrospinal fluid, the pretreatment method is: adding RNase inhibitor to the cerebrospinal fluid.

[0008] Furthermore, when the body fluid is cerebrospinal fluid, the RNase inhibitor is RNase Inhibitor.

[0009] Furthermore, in step (1), the centrifugal temperature is 4°C, the rotation speed is 2000-3000 rpm, and the centrifugal time is 10-15 min.

[0010] Furthermore, in step (2), the adsorption column is a high-quality imported ion membrane that can efficiently separate RNA.

[0011] Furthermore, in step (2), the centrifugal adsorption is carried out at a centrifugal temperature of 4°C, a rotation speed of 11000-13000 rpm, and a centrifugal time of 0.5-2 min.

[0012] Furthermore, in step (2), the step of eluting with the eluent is: (1) Place the adsorption column back into the collection tube and centrifuge at 11,000-13,000 rpm for 2-5 min. Remove the adsorption column and place it into a centrifuge tube. Add 20-50 μL of 65°C preheated eluent and let stand for 1-5 min. Centrifuge at 12,000 rpm and 4°C for 1-5 min to obtain the cfRNA solution.

[0013] The present invention also provides a method for extracting cfRNA from body fluids to obtain the cfRNA.

[0014] The beneficial effects of the present invention compared with the prior art are: (1) The present invention utilizes anhydrous ethanol, ammonium acetate, and linearized acrylamide to prepare a cfRNA precipitant. The cfRNA precipitant of the present invention has a high affinity for cfRNA, but has a low adsorption to proteins and DNA. The cfRNA precipitant of the present invention is used to extract cfRNA from body fluids, and the obtained cfRNA has high purity and high quality.

[0015] (2) The body fluid cfRNA extraction method provided by the present invention can be adapted to a variety of complex biological fluid samples, providing strong technical support for liquid biopsy and disease diagnosis, which can improve the yield and purity of cfRNA, reduce sample processing time, enhance operational convenience, and ensure the molecular integrity of cfRNA for accurate downstream analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0017] Figure 1 This is a flow chart of extracting cfRNA from body fluids according to the present invention; Figure 2 This is a graph showing the concentration and purity of plasma cfRNA extraction and the residual rate of protein and organic matter in Experimental Example 1; Figure 3 This is the Agilent2100 test result diagram for plasma cfRNA extraction in Experimental Example 1; Figure 4 This is a graph showing the test results of serum cfRNA extraction concentration and purity as well as protein and organic matter residual rates in Experimental Example 1; Figure 5 This is the result of Agilent2100 test for serum cfRNA extraction in Experimental Example 1; Figure 6 This is a graph showing the concentration and purity of cfRNA extracted from cerebrospinal fluid and the residual rate of protein and organic matter in Experimental Example 2; Figure 7 This is the Agilent2100 test result diagram for cerebrospinal fluid cfRNA extraction in Experimental Example 2. DETAILED DESCRIPTION

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0020] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0021] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0022] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0023] The present invention provides two methods for extracting cfRNA from body fluids using cfRNA precipitants ( Figure 1 ): 1. When the body fluid is serum or plasma, the method for extracting cfRNA is: (1) Treating the blood with plasma or serum to obtain serum or plasma, adding RNA carrier and mixing evenly, then adding lysis solution and digestion solution, mixing evenly, and heating in a water bath to obtain a mixed solution; (2) adding a precipitant to the mixed solution for precipitation, using an adsorption column for centrifugal adsorption, washing the adsorption column with a washing solution, and eluting with an eluent to obtain a cfRNA solution.

[0024] 2. When the body fluid is cerebrospinal fluid, the method for extracting cfRNA is: (1) Add RNase inhibitor to the cerebrospinal fluid and centrifuge, then add RNA carrier and mix evenly, then add lysis buffer and digestion buffer, mix evenly, and heat in a water bath to obtain a mixed solution; (2) adding a precipitant to the mixed solution for precipitation, using an adsorption column for centrifugal adsorption, washing the adsorption column with a washing solution, and eluting with an eluent to obtain a cfRNA solution. Example 1

[0025] Embodiment 1 of the present invention provides a method for extracting cfRNA from a plasma / serum sample, which specifically comprises the following steps: 1) Blood in EDTA tubes (BD #366643) was used for plasma treatment, and blood in regular serum tubes or rapid serum tubes was used for serum treatment. They were stored at room temperature to obtain plasma / serum samples. The plasma / serum samples were centrifuged at 4°C, 3000 rpm for 10 min to remove the cell pellet.

[0026] 2) Take an RNase-free 1.5 mL centrifuge tube, add 200 μL of centrifuged plasma / serum sample, take 4 μL of RNA Carrier from the Starvio Plasma Free RNA Extraction Kit (N11005:50T) and mix evenly, then add 200 μL of lysis buffer and 20 μL of digestion buffer in the Starvio Plasma Free RNA Extraction Kit, shake and mix, and place in a 65°C water bath for 10 minutes.

[0027] 3) Add 0.9 mL of anhydrous ethanol to the solution after water bath in step 2), add 40 μL of 5 M ammonium acetate (NH4OAc) to make the final concentration 20 μg / mL, add 4 μL of 5 mg / mL linear acrylamide solution (10408ES03) for co-precipitation, gently invert to mix, if there is translucent suspension, it will not affect RNA extraction and subsequent experiments.

[0028] 4) Place the adsorption column in the kit into the collection tube, transfer 700 μL of the solution in step (2) into the adsorption column, centrifuge at 12,000 rpm, 4°C for 1 minute, and discard the waste liquid in the tube.

[0029] 5) Place the adsorption column back into the collection tube, transfer all the remaining solution into the adsorption column, and repeat step 3).

[0030] 6) Place the adsorption column back into the collection tube, add 500 μL of washing solution A in the Starvio plasma free RNA extraction kit (add anhydrous ethanol at a final concentration of 30%) to the adsorption column, let it stand for 2 minutes, centrifuge at 12,000 rpm, 4°C for 1 minute, and discard the waste liquid in the tube.

[0031] 7) Place the adsorption column back into the collection tube, add 500 μL of washing solution B in the Starvio plasma free RNA extraction kit (add anhydrous ethanol at a final concentration of 70%) to the adsorption column, centrifuge at 12,000 rpm and 4°C for 1 minute, and discard the waste liquid in the tube.

[0032] 8) Place the adsorption column back into the collection tube, centrifuge at 12,000 rpm for 3 minutes, and discard the remaining washing solution. At the same time, take 30 μL of the eluate for every 200 μL of plasma / serum sample, place it in a sterile 1.5mL centrifuge tube, and preheat it at 65℃ for 2 minutes.

[0033] 9) Remove the adsorption column and place it in a new 1.5 ml centrifuge tube without RNAse. Add 30 μL of the preheated elution solution mentioned above, let it stand for 2 minutes, and centrifuge at 12,000 rpm and 4°C for 2 minutes to collect the plasma cfRNA solution.

[0034] 10) Store at -80℃ or use directly in the next experiment. Test Example 1

[0035] In Experimental Example 1 of the present invention, the plasma cfRNA and serum cfRNA separated in Example 1 and Example 2 were tested for purity and yield by OneDrop, and the integrity by Agilent 2100. The specific experimental steps are as follows: 1. OneDrop (ultra-micro volume UV-visible spectrophotometer, Od1000+) detects the concentration and purity of isolated RNA: (1) Before OneDrop measurement, the RNA product must be shaken and mixed, and then centrifuged briefly before measurement to ensure the authenticity of the data.

[0036] (2) Blank was performed using the RNase-free ddH2O used to elute the RNA product in this quality control experiment.

[0037] (3) After Blank, RNase-free ddH2O should be measured first, and the value should be between -0.1 and 0.1, and then the RNA product should be measured.

[0038] (4) Take 2 μL of RNA product for measurement each time.

[0039] 2. Agilent 2100 detects the integrity of isolated RNA: Equipment: Agilent 2100; Take 1 μL of serum cfRNA extraction product and use Agilent RNA 6000 Pico kit for analysis time of 3-5 minutes.

[0040] Result analysis: Figure 2 and Figure 4 The cfRNA precipitant of the present invention is shown to improve the concentration and purity of cfRNA extracted from plasma and serum. The purity of nucleic acid is generally judged based on the A260 / A280 ratio. A260 / A280 refers to the ratio of the absorbance of DNA or RNA at 260nm to the absorbance of protein at 280nm, which is used to indicate the purity of DNA or RNA. The A260 / A280 of pure RNA is 2.0; the A260 / A230 of pure RNA is between 1.8 and 2.0. When the sample is contaminated by protein or DNA, the A260 / A280 value will decrease. If the RNA sample is contaminated by phenol, sugars, or lipids, the A260 / A230 value will decrease.

[0041] This result shows that this method significantly assists in enriching cfRNA, and the product contains fewer residual impurities (proteins, pigments, lipids, etc.).

[0042] Figure 3 and Figure 5 The integrity of plasma cfRNA and serum cfRNA was tested using Agilent 2100. Overall, the fragments were distributed between 150 and 200 nt, which is consistent with the fragment characteristics of cfRNA. Example 2

[0043] Embodiment 2 of the present invention provides a method for extracting cfRNA from a cerebrospinal fluid sample, which specifically comprises the following steps: 1) Add 5 μL 40U / μL RNase Inhibitor (R0102-2kU, Bio-Tech) to the obtained cerebrospinal fluid sample and centrifuge at 4°C, 3000rpm for 10 min. Remove obvious cell debris or turbid precipitate, and take the supernatant and freeze it at -80°C.

[0044] 2) Take an RNase-free 1.5 mL centrifuge tube, add 200 μL of the centrifuged cerebrospinal fluid sample, take 4 μL of RNA Carrier from the Starvio Plasma Free RNA Extraction Kit (N11005:50T) and mix evenly, then add 200 μL of lysis buffer and 20 μL of digestion buffer in the Starvio Plasma Free RNA Extraction Kit, shake and mix, and place in a 65°C water bath for 10 minutes.

[0045] 3) Add 0.9 mL of anhydrous ethanol, add 40 μL of 5 M ammonium acetate (NH4OAc) to make the final concentration 20 μg / mL, add 4 μL of 5 mg / mL linear acrylamide solution (10408ES03) for co-precipitation, gently invert to mix, if there is translucent suspension, it will not affect RNA extraction and subsequent experiments.

[0046] 4) Place the adsorption column into the collection tube, transfer 700 μL of the above solution into the adsorption column, centrifuge at 12,000 rpm, 4°C for 1 minute, and discard the waste liquid in the collection tube.

[0047] 5) Place the adsorption column back into the collection tube, transfer all the remaining solution into the adsorption column, and repeat step 3).

[0048] 6) Put the adsorption column back into the collection tube, add 500 μL of washing solution A in the Starvio plasma free RNA extraction kit (add anhydrous ethanol at a final concentration of 30%) to the adsorption column, let it stand for 2 minutes, centrifuge at 12,000 rpm, 4°C for 1 minute, and discard the waste liquid in the collection tube.

[0049] 7) Put the adsorption column back into the collection tube, add 500 μL of washing solution B (add anhydrous ethanol at a final concentration of 70%) in the Starvio plasma free RNA extraction kit into the adsorption column, centrifuge at 12,000 rpm and 4°C for 1 minute, and discard the waste liquid in the collection tube.

[0050] 8) Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm for 3 minutes to remove the remaining washing solution. At the same time, take 30 μL of the eluate from each sample, place it in a sterile 1.5 mL centrifuge tube, and preheat at 65°C for 2 minutes.

[0051] 9) Remove the adsorption column and place it in a new 1.5 ml centrifuge tube without RNAse. Add 30 μL of the preheated elution buffer mentioned above, let it stand for 2 minutes, and centrifuge at 12,000 rpm and 4°C for 2 minutes to collect the cerebrospinal fluid cfRNA solution.

[0052] 10) Store at -80℃ or use directly in the next experiment. Test Example 2

[0053] Experimental Example 2 of the present invention adopts the method of Experimental Example 1, and the RNA isolated in Example 2 is tested for purity by OneDrop, and the yield and integrity are tested by Agilent 2100. The difference from Experimental Example 1 is that the RNA source of this application is cerebrospinal fluid. The results are as follows Figure 6 and Figure 7 shown.

[0054] like Figure 6 As shown in the figure, the use of [extraction reagent N11005:50T + ammonium acetate precipitation + linear acrylamide precipitation aid] can significantly improve the concentration and purity of cfRNA in cerebrospinal fluid. This result shows that after using this method, cfRNA is significantly assisted in enrichment, and the product contains fewer residual impurities (proteins, pigments, lipids, etc.). Since most of the cerebrospinal fluid is an ultrafiltrate of plasma, there are mechanical and permeability barriers between the cerebrospinal fluid and the brain, so the yield of free RNA extracted from it is negligible and the degree of degradation is serious. Therefore, compared with the concentration and purity of plasma extraction, the effect in cerebrospinal fluid is not so significant. Figure 7 As shown, the fragment size is generally low.

[0055] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A cfRNA precipitant, characterized in that The cfRNA precipitant is composed of anhydrous ethanol, ammonium acetate, and linear acrylamide in a volume ratio of 225-400:10-20:

1.

2. A method for extracting cfRNA from body fluid using the cfRNA precipitant according to claim 1, characterized in that: The steps include: (1) After pretreatment of the body fluid, centrifuge and continue to add RNA carrier to mix evenly, then add lysis buffer and digestion buffer, mix evenly, and heat in a water bath to obtain a mixed solution; (2) adding a cfRNA precipitant to the mixed solution for precipitation, using an adsorption column for centrifugal adsorption, washing the adsorption column with a washing solution, and eluting with an eluent to obtain a cfRNA solution.

3. The method for extracting cfRNA from body fluid according to claim 2, characterized in that: In step (1): When the body fluid is serum or plasma, the pretreatment method is: subjecting blood to serum treatment or plasma treatment to obtain serum or plasma; When the body fluid is cerebrospinal fluid, the pretreatment method is: adding RNase inhibitor to the cerebrospinal fluid.

4. The method for extracting cfRNA from body fluid according to claim 3, characterized in that: When the body fluid is cerebrospinal fluid, the RNase inhibitor is RNase Inhibitor.

5. The method for extracting cfRNA from body fluid according to claim 2, characterized in that: In step (1), the centrifugal temperature is 4°C, the rotation speed is 2000-3000 rpm, and the centrifugal time is 10-15 min.

6. The method for extracting cfRNA from body fluid according to claim 2, characterized in that: In step (2), the adsorption column is a high-quality imported ion membrane that can efficiently separate RNA.

7. The method for extracting cfRNA from body fluid according to claim 2, characterized in that: In step (2), the centrifugal adsorption is carried out at a centrifugal temperature of 4°C, a rotation speed of 11000-13000 rpm, and a centrifugal time of 0.5-2 min.

8. The method for extracting cfRNA from body fluid according to claim 2, characterized in that: In step (2), the step of eluting with the eluent is: (1) Place the adsorption column back into the collection tube, centrifuge at 11,000-13,000 rpm for 2-5 min, remove the adsorption column, place it into a centrifuge tube, add 20-50 μL of 65°C preheated eluent, let stand for 1-5 min, and centrifuge at 12,000 rpm at 4°C for 1-5 min to obtain the cfRNA solution.

9. cfRNA extracted according to the method for extracting cfRNA from body fluids as claimed in any one of claims 2 to 8.

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