CN114517196A - Extraction method and application of plasma free miRNA - Google Patents

Abstract

A method for extracting miRNA from a plasma sample and a corresponding kit.

Description

A kind of plasma free miRNA extraction method and its application

technical field

The present invention relates to a method for extracting miRNA from plasma samples and a corresponding kit.

Background technique

MicroRNAs (miRNAs) are a class of endogenous small RNAs of about 18-25 nucleotides in length, which have a variety of important regulatory roles in cells. Each miRNA can have multiple target genes, and several miRNAs can also regulate the same gene. This complex regulatory network can not only regulate the expression of multiple genes through one miRNA, but also fine-tune the expression of a certain gene through the combination of several miRNAs. It is speculated that miRNAs regulate one-third of human genes. Recent studies have shown that about 70% of mammalian miRNAs are located in TUs (transcription units, TUs), and most of them are located in introns. The positions of some intronic miRNAs are highly conserved across species. miRNAs are not only conserved in gene position, but also show a high degree of homology in sequence.

Studies have shown that the expression level of miRNA is closely related to the occurrence of various tumors. It can be used as a tumor suppressor gene to down-regulate the activity of proto-oncogene, and it can also be used as an oncogene to down-regulate the activity of tumor suppressor gene. Among them, the miRNA present in the plasma is also called circulating miRNA, and its source is not completely clear, but may originate from the active secretion of tissue cells. Compared with other tumor detection methods, plasma miRNAs have higher specificity and can clearly distinguish normal people from tumor patients; the detection method is simple, easy to implement and low cost. Plasma miRNAs have tumor markers involved in various stages of tumorigenesis and metastasis, and there are corresponding changes in gene expression at each stage, as well as changes in miRNAs that regulate these genes. The same tumor has different characteristics in different stages of occurrence and development. miRNA expression profile; in addition, different tumors have their own specific miRNA expression profiles, which can reflect the differentiation and development status of tumor tissue, and better monitor and track disease development.

Regarding the detection technology of miRNA, the commonly used methods are fluorescence quantitative PCR, Northern blot, gene chip, digital PCR, high-throughput sequencing, etc. Compared with the detection technology, the extraction method of miRNA is too simple. The commonly used method is still Trizol-based phenol-chloroform extraction. Compared with column separation, Trizol method basically follows the separation technology of mRNA, but this method does not take into account the difference between miRNA and mRNA, for example, the length of miRNA is much smaller than that of mRNA, and miRNA is mainly in the form of protein complex in cells. exist. Therefore, the key to improve the extraction rate of miRNA lies in the separation of miRNA and related proteins.

In recent years, based on miRNA extraction methods, many researchers have done research, using SDS and proteinase K to process proteins in samples by heating. The heating time of this method is too long, and the long-term heating of the sample will definitely lead to partial degradation of miRNA, which will affect the final extraction efficiency. Heating the protease at an excessively high temperature also greatly reduces the activity of the protease, which is not conducive to the pretreatment of the protein in the plasma sample. At present, there is no relatively fast and efficient method for miRNA extraction. Therefore, there is an urgent need to develop an efficient and accurate plasma miRNA extraction method with high extraction rate.

SUMMARY OF THE INVENTION

Specifically, the present invention solves the technical problems existing in the prior art through the following technical solutions.

1. A method of extracting miRNA from a plasma sample, comprising:

(1) preparing the plasma sample;

(2) adding a lysis solution and a binding solution to the plasma sample to obtain a first mixed solution;

(3) adding absolute ethanol to the first mixed solution, centrifuging, discarding the supernatant, thereby obtaining the second mixed solution;

(4) adding lysate to the second mixed solution, then adding dehydrated alcohol, and mixing to obtain the third mixed solution;

(5) transferring the third mixed solution to the silicon membrane column to combine the miRNA with the silicon membrane column; optionally, adding a cleaning solution to the silicon membrane column one or more times;

(6) adding DEPC water or nuclease-free water into the silica gel membrane, and eluting the miRNA to obtain the miRNA.

2. The method according to item 1, wherein the lysing solution comprises one or more components selected from Tris-HCl, guanidine isothiocyanate, EDTA, Tween-20, and NP-40.

3. The method according to item 2, wherein the lysate contains 10-100 mM Tris-HCl; preferably, the lysate contains about 50 mM Tris-HCl.

4. The method according to item 2 or 3, wherein the lysate contains 3-6M guanidine isothiocyanate; preferably, the lysate contains about 4M guanidine isothiocyanate.

5. The method according to any one of items 2 to 4, wherein the lysate contains 2-10 mM EDTA; preferably, the lysate contains about 5 mM EDTA.

6. The method according to any one of items 2 to 5, wherein the lysate contains 1-5% Tween-20; preferably, the lysate contains about 4% Tween-20.

7. The method according to any one of items 2 to 6, wherein the lysate contains 2-10% of NP-40; preferably, the lysate contains about 8% of NP-40.

8. The method according to any one of items 2 to 7, wherein the pH of the lysate is 5.5-7.5; preferably, the pH of the lysate is about 6.0.

9. The method according to any one of items 1 to 8, wherein the binding solution comprises one or more substances selected from the group consisting of silicon dioxide, silicon carbide, iron silicide, diatomaceous earth, etc., and/or hydrochloric acid guanidine.

10. The method of item 9, wherein the bonding fluid comprises silicon carbide.

11. The method according to item 9 or 10, wherein the binding solution comprises silica, silicon carbide, iron silicide, or diatomaceous earth with a concentration of 0.1-1 g/ml and/or a particle size of 100 nm-25 μm ; preferably, the concentration is about 0.6 g/ml; preferably, the particle size is about 3 μm.

12. The method according to any one of items 9 to 11, wherein the binding solution comprises guanidine hydrochloride at a concentration of 0.5-2.0M; preferably, the concentration is about 1M.

13. The method according to any one of items 1 to 12, wherein the volume fraction ratio of the plasma sample to the lysate in the step (2) is 1:1 to 1:4; preferably, the The volume fraction ratio is about 1:3.

14. The method according to any one of items 1 to 13, wherein the volume fraction ratio of the plasma sample to the binding solution in the step (2) is 1:1 to 6:4; preferably, the The volume fraction ratio is about 2:1.

15. The method according to any one of items 1 to 14, wherein, before step (3) adds absolute ethanol, the first mixed solution is left to stand for a period of time t1 at a temperature T1 that is 25-70°C, preferably 25-60°C, more preferably 50-60°C, the time t1 is 1-60min, preferably 5-30min, more preferably 10-30min.

16. The method according to any one of items 1 to 15, wherein, after adding the lysate in step (4), after standing for a period of time t2 at a temperature T2, add dehydrated alcohol, and the temperature T2 is 25 -70°C, preferably 25-60°C, more preferably 50-60°C, the time t2 is 1-60min, preferably 5-30min, more preferably 10-30min.

17. The method according to any one of items 1 to 16, wherein the volume ratio of the absolute ethanol to the sample in the step (3) and/or step (4) is 2:1 to 4:1 ; Preferably, the volume ratio is 3:1.

18. The method according to any one of items 1 to 17, wherein the cleaning solution comprises one or more components selected from Tris-HCl, guanidine isothiocyanate, EDTA, and absolute ethanol.

19. The method according to item 18, wherein the washing solution comprises 10-100 mM Tris-HCl; preferably, about 50 mM Tris-HCl.

20. The method of clause 18 or 19, wherein the cleaning solution comprises 1-3M guanidine isothiocyanate; preferably, about 1M guanidine isothiocyanate.

21. The method according to any one of items 18 to 20, wherein the wash solution comprises 2-10 mM EDTA; preferably, about 8 mM EDTA.

22. The method according to any one of items 18 to 21, wherein the cleaning solution comprises 50-80% absolute ethanol; preferably, about 75% absolute ethanol.

23. The method according to any one of items 18 to 22, wherein the pH of the cleaning solution is 6.0-7.5; preferably, the pH is about 7.0; preferably; the pH is about 6.8-7.2.

24. The method according to any one of items 18 to 23, wherein the volume fraction ratio of the plasma sample to the wash solution is 1:2.

25. The method according to any one of items 1 to 24, wherein the concentration (volume percent) of the DEPC water is 0.01%-1%, preferably 0.05%-0.8%, more preferably 0.06% -0.2%.

26. The method according to any one of items 1 to 25, wherein the silicon membrane column is a solution-permeable filter medium made of silicon material or glass fiber.

27. The method according to any one of items 1 to 26, wherein the plasma sample is from a mammal; preferably, the plasma sample is from a human.

28. A kit for extracting miRNA, comprising a lysing solution, a binding solution, and a washing solution.

29. The kit according to item 28, wherein the lysis solution comprises one or more components selected from Tris-HCl, guanidine isothiocyanate, EDTA, Tween-20, and NP-40.

30. The kit according to item 29, wherein the lysate comprises 10-100 mM Tris-HCl; preferably, the lysate comprises about 50 mM Tris-HCl.

31. The kit according to item 29 or 30, wherein the lysate contains 3-6M guanidine isothiocyanate; preferably, the lysate contains about 4M guanidine isothiocyanate.

32. The kit according to any one of items 29 to 31, wherein the lysate contains 2-10 mM EDTA; preferably, the lysate contains about 5 mM EDTA.

33. The kit according to any one of items 29 to 32, wherein the lysate contains 1-5% Tween-20; preferably, the lysate contains about 4% Tween-20.

34. The kit according to any one of items 29 to 33, wherein the lysate contains 2-10% of NP-40; preferably, the lysate contains about 8% of NP-40.

35. The kit according to any one of items 29 to 34, wherein the pH of the lysate is 5.5-7.5; preferably, the pH of the lysate is about 6.0.

36. The kit according to any one of items 28 to 35, wherein the binding solution comprises one or more substances selected from silicon dioxide, silicon carbide, iron silicide, diatomaceous earth, etc., and/or Guanidine hydrochloride.

37. The kit according to item 36, wherein the binding solution comprises silicon carbide.

38. The kit according to item 36 or 37, wherein the binding solution comprises silicon dioxide, silicon carbide, iron silicide, or diatoms with a concentration of 0.1-1 g/ml and/or a particle size of 100 nm-25 μm soil; preferably, the concentration is about 0.6 g/ml; preferably, the particle size is about 3 μm.

39. The kit according to any one of items 36 to 38, wherein the binding solution comprises guanidine hydrochloride at a concentration of 0.5-2.0M; preferably, the concentration is about 1M.

40. The kit according to any one of items 28 to 39, wherein the cleaning solution comprises one or more components selected from Tris-HCl, guanidine isothiocyanate, EDTA, and absolute ethanol.

41. The kit according to item 40, wherein the washing solution comprises 10-100 mM Tris-HCl; preferably, about 50 mM Tris-HCl.

42. The kit according to item 40 or 41, wherein the cleaning solution comprises 1-3M guanidine isothiocyanate; preferably, about 1M guanidine isothiocyanate.

43. The kit according to any one of items 40 to 42, wherein the wash solution comprises 2-10 mM EDTA; preferably, about 8 mM EDTA.

44. The kit according to any one of items 40 to 43, wherein the cleaning solution comprises 50-80% absolute ethanol; preferably, about 75% absolute ethanol.

45. The kit according to any one of items 40 to 44, wherein the pH of the cleaning solution is 6.0-7.5; preferably, the pH is about 7.0; preferably, the pH is about 6.8-7.2.

46. The kit according to any one of items 28 to 45, comprising DEPC water with a concentration (volume percent) of 0.01%-1%, preferably 0.05%-0.8%, more preferably , 0.06%-0.2%.

47. The kit according to any one of items 28 to 46, comprising a silica membrane column, which is a solution-permeable filter medium made of silica material or glass fiber.

Description of drawings

Figure 1 shows the detection results of fluorescence quantitative PCR.

Detailed description of the invention

The invention provides a plasma miRNA extraction method and a related kit.

In a first aspect of the invention, a method for extracting miRNA from plasma is provided, comprising the following steps:

(1) Provide plasma samples;

(2) to the sample described in step (1), add lysing solution and binding solution to obtain the first mixed solution;

(3) adding absolute ethanol to the first mixed solution, centrifuging, discarding the supernatant, thereby obtaining the second mixed solution;

(4) adding a certain amount of lysate to the second mixed solution, then adding absolute ethanol, and mixing to obtain the third mixed solution;

(5) transferring the third mixed solution to a silicon membrane column and centrifuging;

(6) adding cleaning solution to the silicon membrane column, centrifuging;

(7) Repeat the previous step twice;

(8) Centrifuge again to ensure that there is no liquid in the silicon membrane column;

(9) Add DEPC water or nuclease-free water to the silica gel membrane;

(10) Centrifuge and collect the eluate with a clean nuclease-free centrifuge tube;

In some embodiments, in step (2), the lysing solution is added to the sample first, and then the binding solution is added to obtain the first mixed solution.

In some embodiments, in step (2), a binding solution is added to the sample first, and then a lysis solution is added to obtain a first mixed solution.

In some embodiments, the plasma sample is from a human, mammal (eg, mouse, rat, rabbit, dog, sheep, monkey, etc.). In some embodiments, the plasma sample is from a human.

In some embodiments, the miRNA is from a mammal, preferably a human.

In some embodiments, the content of the components in the lysing solution in step (2) is: 10-100mM Tris-HCl, 3-6M guanidine isothiocyanate, 2-10mM EDTA, 1-5% Tween-20 , 2-10% of NP-40, wherein the pH of the lysate is 5.5-7.5;

Preferably, the content of the components in the lysis solution is: 50mM Tris-HCl, 4M guanidine isothiocyanate, 5mM EDTA, 4% Tween-20, 8% NP-40, wherein the pH of the lysis solution is is 6.0.

In some embodiments, the components of the lysate include one or more of the following. In some embodiments, the lysate contains all of the following components:

• In some embodiments, the concentration of Tris-HCl is 10-100 mM. In some embodiments, the concentration of Tris-HCl is about 10 mM, 20 mM, 30 mM, 40 mM, 60 mM, 70 mM, 80 mM, or 90 mM.

• In some embodiments, the concentration of EDTA is 2-10 mM. In some embodiments, the concentration of EDTA is about 2 mM, 3 mM, 4 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM.

• In some embodiments, the concentration of guanidine isothiocyanate is 3-5M. In some embodiments, the concentration of guanidine isothiocyanate is about 3M, 3.5M, 4M, 4.5M, 5M, or 5.5M.

• In some embodiments, the mass concentration of Tween-20 is between 1% and 8%. In some embodiments, the mass concentration of Tween-20 is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, or 8%.

• In some embodiments, the mass concentration of NP-40 is between 5% and 12%. In some embodiments, the mass concentration of NP-40 is about 5%, 6%, 7%, 8%, 9%, 10%, 11%, or 12%.

• In some embodiments, the pH is between 5.5-7.5. In some embodiments, the pH is about 5.5, 5.8, 6.1, 6.4, 6.7, 7.1, or 7.5.

In some embodiments, Tris-HCl can provide a stable buffer at pH 5.0-7.0, so that the nucleic acid phosphate group and the silica membrane are in a charged state, which is conducive to the formation of hydrogen bonds; guanidine isothiocyanate can provide a high-salt environment, Make the denatured nucleic acid show higher thermodynamic stability than when it is correctly folded or in the natural conformation, promote the formation of hydrogen bonds between the phosphate group of the nucleic acid and the silanol group in the silica adsorption column, which is conducive to the adsorption of nucleic acid in The inner surface of the silica column. EDTA is added to chelate divalent metal ions, thereby inhibiting endonuclease activity and achieving the purpose of protecting nucleic acids.

In some embodiments, the step (2) binding solution is one or two of silicon dioxide, silicon carbide, iron silicide, diatomaceous earth, etc., the concentration is 0.1-1 g/ml, and the particle size is 100 nm-25 μm , 0.5-2.0M guanidine hydrochloride;

Preferably, the component content of the binding solution is: 0.6g/mL silicon carbide, particle size is 3μm, 1M guanidine hydrochloride;

In some embodiments, the components of the binding fluid comprise one or more of the following. In some embodiments, the binding fluid comprises all of the following:

• In some embodiments, the mass concentration of silicon carbide is 0.1-1 g/mL. In some embodiments, the mass concentration of silicon carbide is about 0.2 g/mL, 0.3 g/mL, 0.4 g/mL, 0.6 g/mL, 0.7 g/mL, 0.8 g/mL, 0.9 g/mL, or 1 g /mL.

• In some embodiments, the concentration of guanidine hydrochloride is 0.5-3M. In some embodiments, the concentration of guanidine hydrochloride is about 0.5M, 1.0M, 1.5M, 2M, 2.5M, or 3M.

Silicon carbide is a dark gray crystalline substance, insoluble in water, acid and alkali. Commercial formulations of silicon carbide are available, where they typically consist of greater than 98% SiC, with minor amounts of carbon (C), silicon (Si), silicon dioxide ( _SiO2 ) and iron (Fe) present. Silicon carbide is also available in various particle sizes or grades.

In some embodiments, the ratio of the amount of plasma added in the step (2) to the lysate by volume fraction is 1:1-1:4.

Preferably, the amount of plasma added to the lysate is 1:3 in volume fraction ratio.

In some embodiments, the amount of plasma added in the step (2) and the binding solution are in a volume fraction ratio of 1:1-6:1.

Preferably, the amount of plasma added to the binding solution is 2:1 in volume fraction ratio.

In some embodiments, in the step (3), before adding anhydrous ethanol, the first mixed solution is allowed to stand at a temperature T1 for a period of time t1. In some embodiments, the standing temperature T1 is 25-70°C, preferably 25-60°C, more preferably 50-60°C. In some embodiments, the standing time t1 is 1-60 min, preferably 5-30 min, more preferably 10-30 min.

In some embodiments, in the step (3), the amount of the absolute ethanol is 0.4-1 mL, preferably 0.5-0.8 mL, more preferably 0.7 mL.

In some embodiments, in the step (3), the centrifugation refers to centrifugation at 1250rpm for 30-60s;

In some embodiments, in the step (4), the amount of the lysis solution is 0.1-0.8 mL, preferably 0.2-0.6 mL, more preferably 0.25-0.4 mL.

In some embodiments, in the step (4), after adding the lysing solution, after standing at a temperature T2 for a period of time t2, anhydrous ethanol is added. In some embodiments, the standing temperature T2 is 25-70°C, preferably 25-60°C, more preferably 50-60°C. In some embodiments, the standing time t2 is 1-60 min, preferably 5-30 min, more preferably 10-30 min.

In some embodiments, in the step (3), the anhydrous ethanol is 0.1-0.8 mL, preferably 0.2-0.5 mL, more preferably 0.25-0.4 mL.

In some embodiments, in the step (5), the centrifugation refers to centrifugation at 14000rpm for 1-2min;

In some embodiments, the component content in the cleaning solution in step (6) is: 10-100mM Tris-HCl, 1-3M guanidine isothiocyanate, 2-10mM EDTA, 50-80% absolute ethanol, Wherein, the pH in the cleaning solution is 6.0-7.5;

Preferably, the component contents in the cleaning solution are: 50mM Tris-HCl, 1M guanidine isothiocyanate, 8mM EDTA, 75% absolute ethanol, wherein the pH of the cleaning solution is 7.0;

In some embodiments, the composition of the cleaning solution (wash solution) includes one or more of the following. In some embodiments, the cleaning fluid (washing fluid) comprises all of the following:

• In some embodiments, the concentration of Tris-HCl is 10-100 mM. In some embodiments, the concentration of Tris-HCl is about 10 mM, 20 mM, 30 mM, 40 mM, 70 mM, 80 mM, 90 mM, or 100 mM.

• In some embodiments, the concentration of EDTA is 2-10 mM. In some embodiments, the concentration of EDTA is about 2 mM, 3 mM, 4 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM.

• In some embodiments, the concentration of guanidine isothiocyanate is 0.5-3M. In some embodiments, the concentration of guanidine isothiocyanate is about 0.5M, 1M, 1.5M, 2M, 2.5M, or 3M.

• In some embodiments, the mass percentage of ethanol is 60-80%. In some embodiments, the mass percentage of ethanol is about 60%, 63%, 70%, 75%, or 80%.

• In some embodiments, the pH of the cleaning solution is 6.0-7.5. In some embodiments, the pH of the cleaning solution is about 6.0, 6.2, 6.7, 6.9, 7.0, 7.3, or 7.5.

In some embodiments, the usage amount of the cleaning solution used for cleaning cannot exceed the capacity of the silica gel column, and the usage amount of the cleaning solution is 0.5-0.8 mL.

Preferably, the usage amount of the cleaning solution is 0.6 mL.

In some embodiments, in the step (6), the centrifugation refers to centrifugation at 14000rpm for 1-2min;

In some embodiments, in the step (8), the centrifugation refers to centrifugation at 14000rpm for 2-5min;

In some embodiments, in the step (9), the concentration (volume percentage) of DEPC water is 0.01%-1%, preferably 0.05%-0.8%, more preferably 0.06%-0.2%.

In some embodiments, in the step (9), the amount of DEPC water or nuclease-free water added is 20-100 μL.

In some embodiments, in the step (10), the centrifugation refers to centrifugation at 2000rpm for 1-3min, and then centrifugation at 14000rpm for 2-5min.

In some embodiments, the silicon membrane column refers to a filter medium that is permeable to a solution and is made of silicon material or glass fiber.

The terms "about" or "approximately" in this specification refer to a range (greater or less than) of 10% in either direction of the stated reference value unless otherwise stated or otherwise apparent from the context (eg theoretical limit). is 100%, the range will not exceed 100%).

The advantages of the present invention include, but are not limited to: using commonly used chemical reagents as lysing solution and binding solution of special materials, with the cooperation of other reagents, plasma free RNA and bound protein are separated, free RNA is rapidly and completely released, and Keep the denatured protein in solution. The kit of the invention does not use toxic and odorous reagents, such as phenol chloroform, phenol and other similar reagents, and is less destructive to scientific researchers and the environment. The silica gel column method of the kit of the invention has high efficiency and good quality for free RNA extraction, and can be widely used in downstream genome research, genetic disease research in medicine, gene mutation detection, tumor screening, HLA typing, transplantation matching, etc. ; Judicial paternity test, blood relationship identification, etc.; It is also used in the field of archaeology and biological experiments in universities, middle and primary schools.

specific embodiment

The embodiments of the present invention will be described in detail below with reference to specific examples. The following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.

Experimental materials involved in the examples:

In order to verify the efficiency of extracting small fragments of RNA according to the present invention, the inventors designed a primer probe for microRNA-122, reverse-transcribed the free RNA extracted by the method of the present invention and Qiagen method as a template to synthesize microRNA-122 cDNA, and used Taqman-MGB special fluorescence quantification The microRNA-122 concentration was determined by PCR.

microRNA-122 upstream primer: 5'-GGTTAGTGGAGTGTGACAA-3'

microRNA-122 downstream primer: 5'-CTCAACTGGTGTCGTGGAGT-3'

microRNA-122 reverse transcription primer:

5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAAAACCCCA-3’

microRNA-122 probe sequence: 5'-TTCAGTTGAGAAAACCCCA-3'. In some embodiments, it has a 6-FAM group at the 5' end and an MGB group at the 3' end.

Reagents: Primers and probes were purchased from Shanghai Yingweijieji Technology Trading Co., Ltd., and silica gel adsorption columns were purchased from Hangzhou Laifeng Company. Reverse transcription kit and Taqman-MGB special fluorescence quantitative reaction premix were purchased from Nanjing Novizan Biotechnology Co., Ltd. For details, please refer to the product manual.

The following examples are respectively described from three aspects: preparation of reagents, extraction of nucleic acids, and nucleic acid measurement and detection, wherein Example 1 is the preparation of reagents, Example 2 is the extraction of nucleic acids, and Example 3 is the determination of nucleic acids. At the same time, the Qiagen kit and its method, which have the best nucleic acid extraction effect and the most important extraction effect in the field, are used as a control to describe the effect of nucleic acid extraction using the kit of the present application.

Example 1: Preparation of reagents

1. Preparation of Lysis Solution

50mM Tris-HCl, 4M guanidine isothiocyanate, 5mM EDTA, 4% Tween-20, and 8% NP-40 were prepared by conventional reagent preparation methods, mixed well, and adjusted to pH 6.0. to obtain the lysate.

Wherein, the substance concentration of Tris-HCl is within 10-100mM, for example, it can be: 10mM, 20mM, 30mM, 40mM, 60mM, 70mM, 80mM, or 90mM, etc.;

The amount concentration of EDTA is within 2-10mM, for example, it can be: 2mM, 3mM, 4mM, 6mM, 7mM, 8mM, 9mM, 10mM, etc.;

The substance concentration of guanidine isothiocyanate is within 3-5M, for example, it can be: 3M, 3.5M, 4M, 4.5M, 5M, 5.5M, etc.;

The mass concentration of Tween-20 is within 1%-8%, for example, it can be: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, etc.;

The mass concentration of NP-40 is within 5%-12%, such as: 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, etc.;

The pH is within 5.5-7.5, for example, it can be 5.5, 5.8, 6.1, 6.4, 6.7, 7.1, 7.5, etc.

2. Preparation of binding solution

5g/mL silicon carbide with a particle size of 3μm and 1M guanidine hydrochloride were respectively prepared by a conventional reagent preparation method, and the combined solution was obtained after mixing them uniformly.

The mass concentration of silicon carbide is within 0.1-1g/mL, for example, it can be: 0.2g/mL, 0.3g/mL, 0.4g/mL, 0.6g/mL, 0.7g/mL, 0.8g/mL, 0.9g /mL, 1g/mL, etc.;

The substance concentration of guanidine hydrochloride is within 0.5-3M, for example, it can be: 0.5M, 1.0M, 1.5M, 2M, 2.5M, 3M, etc.

Activation of silicon carbide:

a) Put 6g of silicon carbide 3μm grade resin into a 50ml test tube.

b) Suspend the silicon carbide resin in 15 ml of the desired ultrapure water and vortex.

c) Put it on the aliquot mixer and mix for 30min.

d) Centrifuge at 4000 rpm for 3 minutes to compact the resin particles.

e) Discard the supernatant, add 15 ml of the desired ultrapure water again and vortex, repeat the washing once.

f) Discard the supernatant, re-add 15ml of ultrapure water, mix well, put it into 60°C, and heat for 10min;

g) centrifuge at 4000rpm for 3 minutes to compact the resin particles, discard the supernatant by centrifugation, add 15mL of water and leave overnight;

h) Centrifuge at 4000rpm for 3 minutes to compact the resin particles, and discard the supernatant by centrifugation

i) Add 15 mL of ultrapure water again, mix well, centrifuge at 4000 rpm for 3 minutes, and discard the supernatant;

j) Resuspend the silicon carbide resin in 10 mL of guanidine hydrochloride for later use.

3. Preparation of washing solution

50 mM Tris-HCl, 1 M guanidine isothiocyanate, 8 mM EDTA, 75% absolute ethanol were respectively prepared by conventional reagent preparation methods, and the washing solution was obtained after adjusting the pH to reach 7.0.

Wherein, the substance concentration of Tris-HCl is within 10-100 mM, for example, it can be 10 mM, 20 mM, 30 mM, 40 mM, 60 mM, 70 mM, 80 mM, or 90 mM, etc.;

The amount concentration of EDTA is within 2-10mM, for example, it can be: 2mM, 3mM, 4mM, 6mM, 7mM, 8mM, 9mM, 10mM, etc.;

The amount concentration of guanidine isothiocyanate is within 0.5-3M, for example, it can be: 0.5M, 1M, 1.5M, 2M, 2.5M, 3M, etc.

The mass percentage of ethanol is in the range of 60-80%, such as 60%, 63%, 70%, 75%, 80%, etc.

The pH is within 6.0-7.5, for example, it can be 6.0, 6.2, 6.7, 6.9, 7.0, 7.3, 7.5, etc.

Example 2 Extraction of Nucleic Acids in Plasma

1. Preparation of Plasma Samples

Take fresh EDTA for anticoagulation, centrifuge at 2000g for 15min, and absorb the supernatant; centrifuge the supernatant at 3000g for 15min, absorb the supernatant to obtain plasma, and divide the prepared plasma into 4 equal parts for use.

Among them, 2 parts of the mixed plasma are used to extract free RNA by the method and kit of the present invention, and the remaining 2 parts of the mixed plasma are extracted from the plasma free RNA by Qiagen's miRNeasy Serum/Plasma Advanced Kit.

2. Lysis of Plasma Samples

(1) The first cracking

Take 250 μL of plasma, add 750 μL of lysis solution and 125 μL of binding solution, mix for 30 s, and incubate at 60°C for 30 min.

Wherein, the volume fraction ratio of plasma, lysate and binding solution is 1:1-1:4 for plasma and lysate, and 1:1-6:1 for the volume fraction ratio of plasma and binding solution.

The mixing time is within 30-60s, such as 40s, 50s, 60s, etc.

Among them, the incubation temperature is in the range of 25-70°C, and the incubation time is in the range of 1-60min, for example, the temperature is 25°C, 35°C, 45°C, 55°C, 60°C, 65°C, 70°C, etc. 10min, 20min, 30min, 40min, 50min, 60min, etc.

Wherein, the amount of the added absolute ethanol is 0.4-1 mL, and within this range, for example, the volume is 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1 ml, and the like.

Wherein, the centrifugation is in the range of 30-60s at 1250rpm, for example, the time is 40s, 50s, 60s, etc.

(2) The second cracking

To the pellet after centrifugation, a corresponding volume of lysis buffer was added for the second lysis.

Among them, the amount of lysing solution added again is 0.1-0.8ml, within this range 0.2ml, 0.3ml, 0.4ml, 0.5ml, 0.6ml, 0.7ml, 0.8ml, etc.;

Among them, the incubation temperature is in the range of 25-70°C, and the incubation time is in the range of 1-60min, for example, the temperature is 25°C, 35°C, 45°C, 55°C, 60°C, 65°C, 70°C, etc. 10min, 20min, 30min, 40min, 50min, 60min, etc.

Wherein, the amount of the added absolute ethanol is 0.2-0.8 mL, for example, the volume is 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, and the like.

The mixing time is within 30-60s, such as 40s, 50s, 60s, etc.

3. Adsorption of nucleic acids

The above-treated suspension is transferred to a silica gel adsorption column, and the centrifugation is 14000 rpm for 1-2 min, for example, the time is 60s, 80s, 100s, 120s, etc. Free DNA and RNA (that is, nucleic acid as referred to in the present invention) are adsorbed by the silica membrane under the condition of high salt and low pH value. A high concentration of guanidine salts is a necessary condition for the adsorption of nucleic acids on silica membranes.

4. Washing

To the silica gel column after centrifugation, add 0.5-0.8 mL of washing solution, for example, the volume is 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, etc.

Then, the washing was repeated twice, and after the washing was completed, the silica gel adsorption column was centrifuged at 14,000 rpm for 3 min.

Among them, the rotation speed of centrifugal treatment is in the range of 10000-16000 rpm/min, and the processing time is within 2-5min, for example, the rotation speed is 11000 rpm/min, 12000 rpm/min, 13000 rpm/min, 15000 rpm/min, etc. The duration is 2min, 3min, 4min, 5min, etc.

Washing with a washing solution containing absolute ethanol ensures that the silica gel membrane is clean and free of impurities, and at the same time makes the membrane easy to volatilize and dry.

5. Elution

50 μl of DEPC water was added to the silica gel adsorption column, placed at room temperature for 2 minutes, centrifuged at 1250 rpm/min for 2 minutes to collect the eluate, and then centrifuged at 14,000 rpm/min for 3 minutes to obtain free RNA.

Among them, the rotational speed of the centrifugal treatment is in the range of 1000-1600 rpm, and the processing time is in the range of 1-2 min. The treatment time is 1min, 1.5min, 2min, 3min and so on.

Among them, the rotational speed of the centrifugal treatment is in the range of 10000-16000 rpm, and the processing time is in the range of 1-4min. The treatment time is 1min, 1.5min, 2min, 3min, 4min and so on.

Comparative Example The miRNeasy Serum/Plasma Advanced Kit was used to extract plasma free RNA.

This application uses the miRNeasy Serum/Plasma Advanced Kit to extract plasma free RNA and its kit as a control, and the extraction method refers to the miRNeasy Serum/Plasma Advanced Kit reagent instructions. The specific methods are as follows:

1. Add 60 μL Buffer RPP to 250 μL of plasma, vortex to mix for 20 s, and place at room temperature for 3 min;

2. Centrifuge at 12000g for 3 min, discard the precipitate;

3. Transfer about 230 μL of supernatant to a new centrifuge tube and add 1 volume of isopropanol. Transfer the mixture to the adsorption column, centrifuge for 15s under the condition of centrifugal force greater than 8000g, and discard the effluent;

4. Add 700μL Buffer RWT to the adsorption column, centrifuge for 15s under the condition of centrifugal force greater than 8000g, and discard the effluent;

5. Add 500 μL of absolute ethanol to the adsorption column, centrifuge for 2 min under the condition of centrifugal force greater than 8000 g, and discard the effluent;

6. Transfer the adsorption column to a new centrifuge tube and centrifuge at 14000g for 5min to dry the silica gel membrane;

7. Transfer the adsorption column to a new 1.5 ml centrifuge tube, add 20 μL of nuclease-free water, and centrifuge at 14,000 g for 1 min to collect the eluate to obtain free RNA.

Test example

This test example is an example of the measurement of the free RNA extracted in Example 2 and the comparative example. The RNA is mainly analyzed from two aspects of concentration and qPCR results. The specific operations and test results are as follows:

Concentration analysis

Taking the free RNA extracted by the method of the present invention and the Qiagen method as the object, the extracted free RNA was quantified by using the Qubit quantitative kit.

The experimental results are shown in Table 1:

Table 1

Using the free RNA extracted by the method of the present invention and Qiagen method as a template, microRNA-122 cDNA was synthesized by reverse transcription, and the concentration of microRNA-122 was determined by Taqman-MGB special fluorescent quantitative PCR.

Among them, Taqman-MGB special fluorescent quantitative PCR reaction system is shown in Table 2.

Table 2

component 1T 2x Taq Pro HS Universal Probe Master Mix 10μL Primer F (10μM) 0.4μL Primer-R (10μM) 0.4μL TaqMan probe (10μM) 0.2μL template 2μL ddH₂O 7μL total capacity 20μL

Among them, the above PCR reaction was carried out in a fluorescence quantitative PCR instrument 7500 (ABI company), and the reaction conditions were: 95 °C pre-denaturation for 30 seconds; 95 °C denaturation for 10 seconds, 60 °C annealing for 30 seconds, a total of 45 cycles;

The experimental results are shown in Table 3 and Figure 1.

table 3

Our sample 1 Our sample 2 qiagen sample 1 qiagen sample 2 Ct value 27.051 26.751 28.046 28.116

Although the claims have been set forth in this application as specific combinations of features, it is to be understood that the scope of the present disclosure also includes any novel feature or any combination of novel features or any generalization thereof explicitly or implicitly disclosed herein, whether or not Whether it relates to the same invention as presently claimed in any claim and whether it mitigates any or all of the same technical problems as this invention. The applicant hereby provides notice that new claims may be formulated to such features and/or combinations of features during prosecution of this application or any further application derived therefrom.

Although some embodiments have been shown and described, those skilled in the art will understand that changes may be made to these embodiments without departing from the principles of the invention, the scope of which is defined in the claims.

Those skilled in the art will understand that the components, methods, steps, structures, movements, and cooperation described in this application may be modified (added and/or removed) without departing from the full scope and spirit of the present invention. The scope and spirit covers such modifications and any and all equivalents thereof.

Claims (47)

1. A method of extracting miRNA from a plasma sample, comprising:

(1) preparing the plasma sample;

(2) adding a lysis solution and a binding solution into the plasma sample to obtain a first mixed solution;

(3) adding absolute ethyl alcohol into the first mixed solution, centrifuging, and removing supernatant to obtain a second mixed solution;

(4) adding a lysis solution into the second mixed solution, adding absolute ethyl alcohol, and mixing to obtain a third mixed solution;

(5) transferring the third mixed solution into a silicon membrane column to bind miRNA to the silicon membrane column; optionally, adding a cleaning solution to the silicon membrane column one or more times;

(6) Adding DEPC water or nuclease-free water into the silica gel membrane, and eluting the miRNA to obtain the miRNA.

2. The method of claim 1, wherein the lysate comprises one or more components selected from Tris-HCl, guanidinium isothiocyanate, EDTA, tween-20, NP-40.

3. The method of claim 2, wherein the lysis solution comprises 10-100mM Tris-HCl; preferably, the lysate comprises about 50mM Tris-HCl.

4. The method of claim 2 or 3, wherein the lysis solution comprises 3-6M guanidinium isothiocyanate; preferably, the lysis solution comprises about 4M guanidinium isothiocyanate.

5. The method of any one of claims 2 to 4, wherein the lysis solution comprises 2-10mM EDTA; preferably, the lysis solution comprises about 5mM EDTA.

6. The method according to any one of claims 2 to 5, wherein the lysis solution comprises 1-5% tween-20; preferably, the lysate comprises about 4% tween-20.

7. The method of any one of claims 2 to 6, wherein the lysate comprises 2-10% NP-40; preferably, the lysate contains about 8% NP-40.

8. The method of any one of claims 2 to 7, wherein the lysate has a pH of 5.5-7.5; preferably, the lysate has a pH of about 6.0.

9. The method according to any one of claims 1 to 8, wherein the binding liquid comprises one or more substances selected from silica, silicon carbide, iron silicide, diatomaceous earth, and the like, and/or guanidine hydrochloride.

10. The method of claim 9, wherein the bonding liquid comprises silicon carbide.

11. The method of claim 9 or 10, wherein the binding liquid comprises silica, silicon carbide, iron silicide, or diatomaceous earth at a concentration of 0.1-1g/ml and/or a particle size of 100nm-25 μ ι η; preferably, the concentration is about 0.6 g/ml; preferably, the particle size is about 3 μm.

12. The method of any one of claims 9 to 11, wherein the binding solution comprises guanidine hydrochloride at a concentration of 0.5-2.0M; preferably, the concentration is about 1M.

13. The method according to any one of claims 1 to 12, wherein the ratio of the volume fraction of the plasma sample to the lysate in step (2) is from 1:1 to 1: 4; preferably, the volume fraction ratio is about 1: 3.

14. The method according to any one of claims 1 to 13, wherein the ratio of the volume fraction of the plasma sample to the binding solution in step (2) is 1:1 to 6: 4; preferably, the volume fraction ratio is about 2: 1.

15. The process according to any one of claims 1 to 14, wherein the first mixture is allowed to stand at a temperature T1, T1, preferably 25-60 ℃, more preferably 50-60 ℃, for a time T1 before adding absolute ethanol in step (3), the time T1 being 1-60min, preferably 5-30min, more preferably 10-30 min.

16. The process according to any one of claims 1 to 15, wherein after addition of the lysis solution in step (4), after standing at a temperature T2 for a period of time T2, anhydrous ethanol is added, said temperature T2 being 25-70 ℃, preferably 25-60 ℃, more preferably 50-60 ℃, and said time T2 being 1-60min, preferably 5-30min, more preferably 10-30 min.

17. The method of any one of claims 1 to 16, wherein the volume ratio of the absolute ethanol to the sample in step (3) and/or step (4) is 2:1 to 4: 1; preferably, the volume ratio is 3: 1.

18. The method of any one of claims 1 to 17, wherein the wash solution comprises one or more components selected from Tris-HCl, guanidinium isothiocyanate, EDTA, and absolute ethanol.

19. The method of claim 18, wherein the wash solution comprises 10-100mM Tris-HCl; preferably, it comprises about 50mM Tris-HCl.

20. The method of claim 18 or 19, wherein the cleaning solution comprises 1-3M guanidinium isothiocyanate; preferably, about 1M guanidinium isothiocyanate is included.

21. The method of any one of claims 18 to 20, wherein the wash solution comprises 2-10mM EDTA; preferably, about 8mM EDTA is included.

22. A method according to any one of claims 18 to 21, wherein the cleaning solution comprises 50-80% absolute ethanol; preferably, it comprises about 75% absolute ethanol.

23. A method according to any one of claims 18 to 22, wherein the pH of the wash liquid is 6.0-7.5; preferably, the pH is about 7.0; preferably; the pH is about 6.8-7.2.

24. The method of any one of claims 18 to 23, wherein the volume fraction ratio of the plasma sample to the wash solution is 1: 2.

25. The method according to any one of claims 1 to 24, wherein the DEPC water is present in a concentration (volume%) of 0.01-1%, preferably 0.05-0.8%, more preferably 0.06-0.2%.

26. The method of any one of claims 1 to 25, wherein the silicon membrane column is a solution-permeable filter medium made of a silicon material or glass fibers.

27. The method of any one of claims 1 to 26, wherein the plasma sample is from a mammal; preferably, the plasma sample is from a human.

28. A kit for extracting miRNA comprises lysis solution, binding solution and cleaning solution.

29. The kit of claim 28, wherein the lysis solution comprises one or more components selected from Tris-HCl, guanidinium isothiocyanate, EDTA, tween-20, NP-40.

30. The kit of claim 29, wherein the lysate comprises 10-100mM Tris-HCl; preferably, the lysate comprises about 50mM Tris-HCl.

31. The kit of claim 29 or 30, wherein the lysis solution comprises 3-6M guanidinium isothiocyanate; preferably, the lysis solution comprises about 4M guanidinium isothiocyanate.

32. The kit of any one of claims 29 to 31, wherein the lysis solution comprises 2-10mM EDTA; preferably, the lysis solution comprises about 5mM EDTA.

33. The kit according to any one of claims 29 to 32, wherein the lysis solution comprises 1-5% tween-20; preferably, the lysate comprises about 4% tween-20.

34. The kit of any one of claims 29 to 33, wherein the lysis solution comprises 2-10% NP-40; preferably, the lysate contains about 8% NP-40.

35. The kit of any one of claims 29 to 34, wherein the lysate has a pH of 5.5-7.5; preferably, the lysate has a pH of about 6.0.

36. The kit according to any one of claims 28 to 35, wherein the binding liquid comprises one or more substances selected from silica, silicon carbide, iron silicide, diatomaceous earth, and the like, and/or guanidine hydrochloride.

37. The kit of claim 36, wherein the binding liquid comprises silicon carbide.

38. The kit according to claim 36 or 37, wherein the binding liquid comprises silica, silicon carbide, iron silicide, or diatomaceous earth at a concentration of 0.1-1g/ml and/or a particle size of 100nm-25 μ ι η; preferably, the concentration is about 0.6 g/ml; preferably, the particle size is about 3 μm.

39. The kit of any one of claims 36 to 38, wherein the binding solution comprises guanidine hydrochloride at a concentration of 0.5-2.0M; preferably, the concentration is about 1M.

40. The kit of any one of claims 28 to 39, wherein the wash solution comprises one or more components selected from Tris-HCl, guanidinium isothiocyanate, EDTA, and absolute ethanol.

41. The kit of claim 40, wherein the wash solution comprises 10-100mM Tris-HCl; preferably, it comprises about 50mM Tris-HCl.

42. The kit of claim 40 or 41, wherein the wash solution comprises 1-3M guanidinium isothiocyanate; preferably, about 1M guanidinium isothiocyanate is included.

43. The kit of any one of claims 40 to 42, wherein the wash solution comprises 2-10mM EDTA; preferably, about 8mM EDTA is included.

44. The kit of any one of claims 40 to 43, wherein the wash solution comprises 50-80% absolute ethanol; preferably, it comprises about 75% absolute ethanol.

45. The kit according to any one of claims 40 to 44, wherein the pH of the wash solution is 6.0-7.5; preferably, the pH is about 7.0; preferably, the pH is about 6.8 to 7.2.

46. The kit according to any one of claims 28 to 45, comprising DEPC water at a concentration (volume%) of 0.01% -1%, preferably 0.05% -0.8%, more preferably 0.06% -0.2%.

47. A kit according to any one of claims 28 to 46 comprising a silica membrane column, said silica membrane column being a solution permeable filter medium made from a silica material or glass fibres.

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