阅读说明：本技术 一种体外合成siRNA的纯化方法 (Purification method for in vitro synthesis of siRNA ) 是由 李萍 唐雪明 李学文 于 2021-10-12 设计创作，主要内容包括：本发明公开了一种体外合成siRNA的纯化方法。本发明的纯化方法具体指体外转录合成siRNA后进行纯化,得到高纯度的siRNA。具体操作流程：将带有T7启动子的DNA模板体外转录合成siRNA；DNaseI降解DNA模板,S1核酸酶降解单链DNA和RNA；磁珠纯化富集siRNA；核酸吸附柱进一步去除小于20nt的核酸、碱基,以得到纯度很高的siRNA。这种制备方式快捷、便宜,且产物的纯度比市场销售的siRNA体外转录试剂盒要高很多,避免了市售体外转录试剂盒得到的产物中有过多杂质,而使实验结果非常不稳定。极大的提高了体外转录制备siRNA的质量,提高实验效率,减少假阴性的实验结果,具有非常高的推广价值。(The invention discloses a purification method for in vitro synthesis of siRNA. The purification method of the invention specifically refers to that after siRNA is synthesized by in vitro transcription, the siRNA is purified to obtain high-purity siRNA. The specific operation flow is as follows: in vitro transcribing the DNA template with the T7 promoter to synthesize siRNA; DNaseI degrades a DNA template, and S1 nuclease degrades single-stranded DNA and RNA; purifying and enriching siRNA by magnetic beads; the nucleic acid adsorption column further removes nucleic acid and base less than 20nt to obtain siRNA with high purity. The preparation method is quick and cheap, the purity of the product is much higher than that of the siRNA in-vitro transcription kit sold in the market, and the phenomenon that the product obtained by the in-vitro transcription kit sold in the market contains too many impurities, so that the experimental result is very unstable is avoided. Greatly improves the quality of siRNA preparation by in vitro transcription, improves the experimental efficiency, reduces the experimental result of false negative, and has very high popularization value.)

1. A method for purifying in vitro synthesized siRNA is characterized by comprising the following steps:

s1, carrying out in vitro transcription on the DNA single strand containing the target gene to obtain an in vitro transcription product containing the target siRNA;

s2, digesting the in-vitro transcription product by DNaseI and S1 nuclease to obtain a digested product;

s3, adding magnetic beads into the digestion product to perform target siRNA enrichment treatment to obtain an enriched product;

s4, purifying the enriched product by adopting a nucleic acid adsorption column to obtain purified siRNA;

wherein, the nucleic acid adsorption column is filled with at least 4 layers of nucleic acid adsorption membranes with the pore diameter of 8-12 nm.

2. The purification method of in vitro synthesized siRNA of claim 1, wherein the nucleic acid adsorption membrane is glass fiber membrane or silica gel membrane.

3. The method of claim 1, wherein the magnetic beads are selected from the group consisting of silicon hydroxyl magnetic beads, carboxyl magnetic beads, and amino magnetic beads.

4. The method of claim 3, wherein the magnetic beads have a particle size of about 0.8-1.2 μm and a surface charge of about-15 mV to-40 mV.

5. The method for purifying in vitro synthetic siRNA of claim 1, wherein DNaseI concentration is 0.3U/. mu.l to 0.4U/. mu.l at the time of digestion treatment.

6. The method for purifying in vitro synthetic siRNA of claim 1, wherein the concentration of S1 nuclease is 0.8U/. mu.l to 1.2U/. mu.l at the time of digestion treatment.

7. The method for purifying in vitro synthesized siRNA of claim 1, wherein when purifying the enriched product by using nucleic acid adsorption column, the method specifically comprises:

s41, loading the enriched product into a nucleic acid adsorption column, and centrifuging to remove waste liquid;

s42, cleaning the sample on the nucleic acid adsorption column by using RPE Solution, and centrifuging to remove liquid on the nucleic acid adsorption column;

s43, dissolving the siRNA sample on the nucleic acid adsorption column by DEPC water to obtain purified siRNA.

8. The method for purifying siRNA synthesized in vitro according to claim 7, wherein in the step S41 or S42, the centrifugation is performed at 10000-14000 rpm for 1-3 min.

9. The method of claim 1 wherein the single strand of DNA containing the target gene further comprises a 5 'enhancer, a T7 promoter and a 3' dTdT.

10. The method for purifying in vitro synthetic siRNA of claim 1, wherein in step S1, the step of obtaining in vitro transcription product containing siRNA of interest by in vitro transcription comprises:

s11, annealing four DNA single strands containing target genes into 2 pairs of double-stranded DNA;

s12, 2 transcribing single-stranded RNA from the double-stranded DNA, wherein one is a sense strand and the other is an antisense strand;

s13, annealing and hybridizing the two single-stranded RNAs to obtain double-stranded RNA which is an in vitro transcription product.

Technical Field

The invention relates to the technical field of siRNA preparation, in particular to a purification method for in vitro synthesis of siRNA.

Background

siRNA technology was discovered in plants over the last century. In recent years, mammals have been increasingly studied because of their potential as a simple and effective genetic tool to replace gene knock-out. It has made important progress in the research of specific gene function in biological genome, blocking and blocking pathogen gene expression, etc. and has shown excellent application foreground. The siRNA technology brings hope in treating various diseases which can not be cured, such as tumor, hepatitis, hereditary disease, and the like, and is also the hot of new drug development at present.

The first non-viral vector administered siRNA drug, Patisiran, developed by Alnylam corporation worldwide was a drug that specifically silences hATTR expression. hATTR is caused by human transthyretin (TTR) amyloid deposition, and is a rare and difficult-to-cure genetic disease. Patisiran forms a complex with liposome, and is administered by intravenous injection to silence the expression of hATR mRNA in human body and reduce the production of transthyretin. To date, Alnylam corporation has also developed the siRNA drugs Givosiran, Iumasiran, Inclisiran.

STP705, a drug in the research on Shengnuo pharmaceuticals, is a siRNA therapy drug. It consists of two siRNAs, targeting mRNA of TGF-beta 1 and COX-2, respectively, and mixed into a nanoparticle formulation by histidine-lysine polypeptide copolymer (HKP). Each siRNA can inhibit the expression of target mRNA, and the combined use of the two siRNAs has the synergistic effect of reducing the profibrosis and the proinflammatory reaction. Preclinical studies of this company have shown that the drug STP705 also has tumor-inhibiting effects.

The current siRNA preparation methods include the following: 1) chemical synthesis method. The method is prepared by a nucleic acid synthesizer, is suitable for short-term in vitro experimental research, and can be used for chemical modification such as sulfo, 2-OMe, 2-F' and the like. Because the chemical synthesis price cost is higher, the selling price of a 2OD 21bp siRNA is about 600 yuan, and if chemical modification is added, the price is higher, so that the siRNA is not suitable for long-term research; 2) plasmid vector synthesis. According to the method, the siRNA sequence is constructed into a plasmid of a T7 or U6 promoter, the plasmid is transferred into a cell, siRNA can be continuously generated in the cell, and the method is suitable for long-term research. The method is expensive, relatively complex in construction process and only suitable for in vitro research; 3) viral vector synthesis. The method can be used for in vivo experiments by integrating siRNA with adenovirus vectors or retrovirus vectors. The method has the advantages of complex construction process, relatively low success rate and potential safety hazard; 4) in vitro transcription methods. The method begins with a DNA single strand with a T7 promoter, takes four NTPs as substrates, anneals into two double-strand DNA as a template, and then adds a T7 enzyme to transcribe the template to obtain the siRNA. In the existing in vitro transcription kits in the market, the obtained product is usually further purified by magnetic beads, which indicates that high-purity siRNA can be obtained. The method has low cost and short time, can be prepared in a short time, and is suitable for in vivo and in vitro experiments. The method has the defects that although target siRNA can be obtained through AKTA equipment analysis, the purity of the product still needs to be further improved, and the method is easy to generate more impurities, such as mismatched short nucleic acid and base, so that the product is not suitable for the research and application of higher-requirement medical grade level. So far, a rapid, efficient and stable method for obtaining high-purity siRNA is still one of the key points in the research and development of nucleic acid biomedicine field.

Disclosure of Invention

In order to solve the problems that the purity of siRNA prepared by the existing in vitro transcription method or a commercially available kit is not high and the subsequent experimental results are influenced, the invention provides a brand-new method for purifying after in vitro transcription synthesis, and aims to obtain a product of high-purity siRNA.

The first purpose of the invention is to provide a purification method for synthesizing siRNA in vitro, which comprises the following steps:

s1, carrying out in vitro transcription on the DNA single strand containing the target gene to obtain an in vitro transcription product containing the target siRNA;

s2, digesting the in-vitro transcription product by DNaseI and S1 nuclease to obtain a digested product;

s3, adding magnetic beads into the digestion product to perform target siRNA enrichment treatment to obtain an enriched product;

s4, purifying the enriched product by adopting a nucleic acid adsorption column to obtain purified siRNA;

wherein, the nucleic acid adsorption column is filled with at least 4 layers of nucleic acid adsorption membranes with the pore diameter of 8-12 nm.

Further, the nucleic acid adsorption membrane is a glass fiber membrane or a silica gel membrane.

Further, the magnetic beads are silicon hydroxyl magnetic beads, carboxyl magnetic beads or amino magnetic beads.

Furthermore, the magnetic beads have the particle size of about 0.8-1.2 mu m and the surface charge of about-15 mV to-40 mV.

Further, the concentration of DNaseI during digestion treatment was 0.3U/. mu.l to 0.4U/. mu.l.

Further, the concentration of S1 nuclease at the time of digestion treatment was 0.8U/. mu.l to 1.2U/. mu.l.

Further, when the enriched product is purified by using a nucleic acid adsorption column, the method specifically comprises the following steps:

s41, loading the enriched product into a nucleic acid adsorption column, and centrifuging to remove waste liquid;

s42, cleaning the sample on the nucleic acid adsorption column by using RPE Solution, and centrifuging to remove liquid on the nucleic acid adsorption column;

s43, dissolving the siRNA sample on the nucleic acid adsorption column by DEPC water to obtain purified siRNA.

Further, in the step S41 or S42, the centrifugation is performed at 10000-14000 rpm for 1-3 min.

Further, in the step S41, Buffer a is added to the enriched product, 70% to 100% ethanol is added, and the mixture is mixed uniformly and then subjected to sample loading.

Further, when the sample on the nucleic acid adsorption column is washed by using the RPE Solution in the step S42, the washing can be repeated 2 to 4 times.

Furthermore, the single-stranded DNA containing the target gene also comprises an enhancer at the 5 'end, a T7 promoter and a dTdT at the 3' end.

Further, the step of obtaining in vitro transcription product containing target siRNA by in vitro transcription comprises:

s11, annealing four DNA single strands containing target genes into 2 pairs of double-stranded DNA;

s12, 2 transcribing single-stranded RNA from the double-stranded DNA, wherein one is a sense strand and the other is an antisense strand;

s13, annealing and hybridizing the two single-stranded RNAs to obtain double-stranded RNA which is an in vitro transcription product.

The invention has the beneficial effects that:

generally, the biological enzymes for hydrolyzing siRNA nonspecific nucleic acid in a commercially available kit are DNaseI and RNase T1, while RNase T1 specifically degrades only single-stranded RNA at G residues, the invention uses DNaseI and S1 nuclease, wherein S1 nuclease is a single-stranded specific endonuclease that can hydrolyze single-stranded RNA or DNA into 5' mononucleotide, and S1 nuclease is used to sufficiently degrade single-stranded DNA and RNA in the crude product, thereby improving the purity of double-stranded siRNA;

after the crude product is treated by the biological enzyme, the purity of the target siRNA is improved to the maximum extent by continuously using two modes of magnetic bead enrichment and adsorption column purification, and particularly, at least more than 4 layers of glass fiber films with the aperture of 10 +/-2 nm are filled in the adsorption column used by the invention, so that single chains, basic groups and mismatched double chains of less than 20nt pass through the adsorption column, and the target siRNA product is combined in the adsorption column; the purity of the double-stranded siRNA is further improved; the conventional method is one of magnetic bead purification and PAGE gel purification, wherein the purified product of the conventional method contains more impurities, and the purified product of the conventional method is time-consuming and labor-consuming and the siRNA is easy to degrade;

the method greatly improves the quality of siRNA preparation by in vitro transcription, improves the experimental efficiency, reduces the experimental result of false negative, and has very high popularization value.

Description of the drawings:

FIG. 1 is an agarose electrophoresis image of siRNA synthesized by a commercial in vitro transcription kit, wherein 1-3 represent siIL-25, siTNF-alpha and siGAPDH, 4 represents the position of bromophenol blue, and 5 represents the position of xylene blue, respectively;

FIG. 2 is a graph of AKTA analysis of siRNA synthesis by a commercially available in vitro transcription kit;

FIG. 3 shows the detection of mRNA levels of target genes by cell transfection after siGAPDH production using a commercially available kit and the method of example 1;

FIG. 4 is a graphic representation of the AKTA analysis using a commercial kit in combination with the ` third step purification ` of example 1;

FIG. 5 is a graph of AKTA analysis of siRNA products after purification by the method of example 1;

FIG. 6 shows the detection of target gene protein levels by cell transfection after preparation of siGAPDH product using commercially available kits and the method of example 1;

fig. 7 is a grey scale statistical plot of protein levels.

Detailed Description

The present invention is further described below in conjunction with specific examples to enable those skilled in the art to better understand the present invention and to practice it, but the examples are not intended to limit the present invention.

Main reagents and equipment: DNA primers were purchased from Huada, T7 RNAi Transcription Kit was purchased from Novozan (mainly using NTP Mix, 10 × Transcription Buffer, T7 Enzyme Mix and magnetic beads therein), DNaseI Enzyme and S1 nuclease were purchased from Sayborar, multiple layers of glass fiber films in a nucleic acid adsorption column were purchased from Mibor, DMEM medium was purchased from Saybolt, serum was purchased from Saybolt, RNA extraction Kit was purchased from Tiangen, reverse Transcription Kit was purchased from Saybolt, Q-PCR Kit was purchased from Stargard, AKTA device information was purchased from GE, fluorescent Q-PCR was purchased from Bio-Rad, GAPDH primary antibody, secondary antibody was purchased from abcam, lip3000 was purchased from Saybolt.

The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1:

this example illustrates the in vitro synthesis of siRNA using siGAPDH, siIL-25 and siTNF- α.

Step I, annealing four single-stranded DNAs into 2 pairs of double-stranded DNAs: template A, template B:

(1) four single-stranded DNA templates were obtained by chemical synthesis, each comprising a 5 '6-base enhancer, a 20-base T7 promoter, 21 target gene bases, and a 3' dTdT. The sequences are shown in table 1 below:

TABLE 1 primer Table involved in vitro siRNA synthesis

The annealing system is as follows in table 2:

TABLE 2 annealing System

(2) And (3) annealing procedure: heating the water bath to 95 deg.C, inserting the pcr tube of the annealing system into the float, and floating in the water bath for 2 min; and then closing the water bath kettle to naturally reduce the water temperature to the room temperature for about 5 hours.

Step two pairs of templates transcribe single-stranded RNA respectively, wherein one is a sense strand and the other is an antisense strand:

the transcription system was configured as follows:

TABLE 3 transcription System

Reagent	Volume of
		Form A	4μl
Form B	4μl
		10×Transcription Buffer	2μl
NTP Mix	8μl
		T7 Enzyme Mix	2μl

Annealing and hybridizing to obtain double chains:

the water bath was heated to 37 ℃ and the pcr tube with the annealing system was inserted into the float and floated in the water bath overnight.

After obtaining the transcription products, the three groups of siRNA are purified by a purification method carried by the kit.

As shown in figure 1, after the in vitro transcription product of the commercial kit is subjected to a purification mode carried by the kit, the in vitro transcription product is subjected to 2% agarose electrophoresis, wherein a Marker in the figure represents DL5000, the second to fourth lanes represent siRNA, 1 to 3 represent the positions of siIL-25, siTNF-alpha and siGAPDH, 4 represents the position of bromophenol blue, and 5 represents the position of xylene cyan respectively. The single band is seen initially, which indicates that the purity of the siRNA product meets the requirement. The 3 siRNA products were analyzed for purity on AKTA instruments (FIG. 2), and the peak areas of the target regions were all smaller than the area of the peak-rich region. Wherein the siIL-25 target peak area accounts for about 29.249%, the siTNF-alpha target peak area accounts for about 20.425%, and the siGAPDH target peak area accounts for about 19.772%, which proves that the actual percentage of siRNA in the solution is lower than 50%, which is far lower than the requirement of the expected high-purity siRNA, and the risk of failure of the subsequent experiment is increased.

Further purification according to the methods of the present application:

step IV, first-step purification: DNaseI degrades DNA templates, S1 nuclease degrades single-stranded DNA and RNA:

the enzyme digestion system was configured as follows:

TABLE 4 enzymatic digestion System

(2) The water bath is heated to 37 ℃, and the pcr tube with the annealing system is inserted into the buoy and floats in the water bath for 30 min.

Step two, purification: magnetic bead purification enrichment of siRNA:

the siRNA was purified using magnetic beads according to the T7 RNAi Transcription Kit instructions.

Step sixthly, purification in the third step: and further removing nucleic acid and base less than 20nt by using the nucleic acid adsorption column to obtain siRNA with high purity:

(1) adding 360 mu l of Buffer A into the siRNA solution to be purified, and uniformly mixing;

(2) adding 600 mul of absolute ethyl alcohol, and uniformly mixing;

(3) placing the nucleic acid adsorption column in a collection tube, transferring all the mixed solution into the nucleic acid adsorption column, centrifuging at 12000rpm for 1min, and discarding the waste solution;

(4) adding 700 μ l RPE Solution, centrifuging at 12000rpm for 1min, and discarding the waste liquid;

(5) adding 300 μ l RPE Solution, centrifuging at 12000rpm for 1min, and discarding the waste liquid;

(6) placing the nucleic acid adsorption column in a collection tube, centrifuging at 12000rpm for 2min, and spin-drying;

(7) and (3) dripping 30 mu l of DEPC water into the center of the adsorption column, standing at room temperature for 3min, centrifuging at 12000rpm for 1min, and obtaining a solution, namely the final purified siRNA.

Since protein expression of GAPDH as a housekeeping gene in the same cell or tissue is generally constant and is referred to in Western blotting (Western blot) and quantitative fluorescence PCR (qPCR), GAPDH gene is very important in the experiment. In the case of siRNA transfection experiments, siGAPDH was usually set as a positive control group.

Figure 4 is a graph of AKTA analysis using a combination of kit plus 'third step purification'. The AKTA analysis result of fig. 4 shows that the peak area of the siRNA target region in 3 is much larger than the area of the hetero peak region after the combined use, wherein the actual purity of siGAPDH is about 80.112%, the purity can be obviously improved, but the hetero peak still exists obviously.

The results of fig. 5 show that the peak areas of the 3 siRNA target regions are much larger than the area of the hetero peak region, wherein the actual purity of siGAPDH is further increased to 91.293%, the hetero peaks are further reduced, and the target peak occupies the major area. Compared with the figure 2, the purity of the target siRNA product is greatly improved, and the stability of subsequent experiments is further ensured.

Example 2:

using the siGAPDH product obtained in example 1 as an example, cells were transfected and the level of mRNA of the target gene was determined.

(1) Cell preparation

HaCat cells were seeded into 12-well plates to give cell densities between 30% and 40% the next day before transfection.

(2) Transfection of siGAPDH

Mu.l of the basal medium was added to 5. mu.l of lip3000 as tube A, 50. mu.l of the basal medium was added to an appropriate amount of siGAPDH (to give a transfection concentration of 25pmol, 50pmol, 100pmol, 150pmol, 200pmol for the kit product, and 25pmol, 50pmol for the product of example 1) as tube B, and tube B was quickly added to tube A and gently mixed, left to stand at room temperature for 10min, and added dropwise to 12-well plates. Each experimental group was run in 3 replicates with the addition of 1 positive control, one liposome control without siRNA, and one cell blank control.

(3) After 48h of cell culture, cells were collected and RNA was extracted

Adding 400 mul Trizol into each hole, repeatedly blowing the solution by a liquid transfer gun for 10 times, and transferring the solution into a 1.5ml EP tube without nuclease;

adding 20 mul of chloroform, shaking and mixing uniformly for 30s, standing for 10min on ice, and centrifuging at 12000rpm for 10min at 4 ℃;

sucking the transparent water phase at the uppermost layer into another EP tube by a small wing;

③ adding 200 mul of isopropanol, mixing evenly, standing for 10min on ice, centrifuging at 12000rpm at 4 ℃ for 10min, discarding the supernatant, and precipitating RNA at the bottom of the cell to be white;

adding 1ml of 75% ethanol (prepared by DEPC water), gently washing the RNA precipitate, centrifuging at the temperature of 4 ℃ at 12000rpm for 5min, and discarding the supernatant as much as possible;

throwing the EP tube empty, centrifuging at 12000rpm at 4 ℃ for 2min, and discarding the supernatant as much as possible to ensure that no macroscopic liquid exists in the EP tube;

sixthly, opening the cover and airing for 10min at room temperature;

seventhly, adding 30 mu l of DEPC water to dissolve the precipitate, and properly placing the precipitate into a water bath kettle at 37 ℃ to heat so as to accelerate the dissolution of the precipitate;

measuring the concentration and the ratio of RNA A260/280 quantitatively.

(4) Reverse transcription of RNA

(ii) reverse transcription of RNA to obtain cDNA according to Table 5 below

TABLE 5 reverse transcription configuration System

Reagent	Volume of
		5×RT Buffer	4μl
M-MLV(H-)Reverse Transcription Mix	1μl
		RNA	1000ng
oligo(dT)20(10μM)	4μl
		DEPC water	Make up to 20. mu.l

Operating a program of the PCR instrument: 42 ℃ for 1 h; 5min at 85 ℃; infinity at 4 ℃;

③ adding 180 mul of purified water into 20 mul of cDNA stock solution, and mixing evenly.

(5) Q-PCR assay

The Q-PCR system was configured to detect the expression of the target gene as follows:

TABLE 6Q-PCR configuration System

Reagent	Volume of
		2×Q-PCR Mix	10μl
cDNA	20ng
		Primer upstream (10. mu.M)	1μl
Primer downstream (10. mu.M)	1μl
		Pure water	Make up to 20. mu.l

Operating a Q-PCR instrument: 3mins at 95 ℃; 39 cycles (95 ℃, 15 s; 60 ℃, 30 s); 15s at 95 ℃; 60 ℃ for 1 min; 95 ℃ for 15 s. Using GAPDH as internal reference, 3 replicates, observing the dissolution curve and CT value, using 2^-△△CtAnd calculating the expression level of the target gene.

(6) Results of the experiment

FIG. 3 shows that siGAPDH was knocked down less efficiently at 24h in the in vitro transcription kit for cellular mRNA levels. After the siRNA is prepared and purified by the method of the embodiment 1, the siRNA knockdown is about 50 percent, and the effect is good. The useful siRNA sequence can obtain better experimental results, and the experimental efficiency and the cost are improved.

Example 3:

using the siGAPDH product obtained in example 1 as an example, cells were transfected and the expression level of the target gene protein was determined.

(1) Cell preparation

HaCat cells were seeded into 12-well plates to give cell densities between 30% and 40% the next day before transfection.

(2) Transfection of siGAPDH

Mu.l of the basal medium is added with 5 mu.l of lip3000 to be used as a tube A, a proper amount of siGAPDH is added into 50 mu.l of the basal medium to be used as a tube B, the tube B is rapidly added into the tube A and is gently mixed, the mixture is kept stand for 10min at room temperature, and the mixture is dripped into a 12-hole plate. The transfection concentrations of the products were 25pmol, 50pmol and 100pmol, respectively. Each experimental group was run in 3 replicates with the addition of 1 positive control, one liposome control without siRNA, and one cell blank control.

(3) Culturing for 48 hr, collecting cells, and extracting protein

Washing cells once with PBS, adding 200ul of cell lysis solution (RIPA) containing protease inhibitor, fully blowing by a pipette, and transferring to a 1.5ml EP tube; centrifuging at 12000rpm for 5min at room temperature, and collecting the cell phone supernatant as the cell total protein product;

② placing into a water bath kettle at 95 ℃ for 5min to ensure that the protein is fully denatured, and storing at-80 ℃ for later use after quantifying the protein.

(4) Western Blot detection

Preparing glue, loading, 60V, 30min, 120V, 90 min;

reducing the PDVF membrane with a moderate size, activating the methanol for 20s, removing the methanol, washing for 5min by pure water, disassembling the running glue, putting the running glue into a washing box of the membrane, removing the water, pouring a membrane transferring solution, and washing for 15min on a shaking table;

thirdly, placing a board on a white board of a film rotating board, placing glue on a black surface, placing glue on the middle layer of cotton, adding glue to 2 layers of cotton and two layers of cotton, and cutting a notch at the upper left corner of the film to mark;

putting the mixture into a film transferring groove, putting the mixture into a basin, pouring the film transferring liquid in the groove, covering the basin with a cover, pouring ice to cover the basin, and putting a proper amount of water (the water does not exceed the cover, so that the current is prevented from being unstable) at 100V for 2 hours;

diluting 5% skimmed milk powder with TBST, placing the membrane into 5% milk container, and sealing with shaking table for 2 h;

sixthly, washing the membrane slightly in TBST, diluting the primary antibody with a primary antibody diluent, coating the membrane and standing at room temperature overnight;

seventhly, washing the membrane by using TBST +0.1 percent Tween-20 at room temperature for 10min for 3 times;

eighthly, diluting the secondary antibody with a secondary antibody diluent, coating a film, and keeping the room temperature for 2 hours;

ninthly, washing the membrane with TBST for 4 times at room temperature, washing the membrane with TBS for 1 time at room temperature for 5 min; and developing in a dark room.

(5) Results of the experiment

FIG. 6 is a Western Blot plot with No. 1, No. 2, No. 3 sinC, i.e., a control sequence without interfering with efficiency; no. 4, No. 5 and No. 6 are siGAPDH synthesized by the kit; no. 7, No. 8, No. 9 are siGAPD synthesized by the present invention; m is marker. Wherein the transfection concentrations No. 1, No. 4, No. 7 were 25 pmol; the transfection concentrations of Nos. 2, 5 and 8 were 50 pmol; the transfection concentrations of No. 3, No. 6 and No. 9 were 100 pmol. The No. 4, No. 5 and No. 6 synthesized by the kit have cytotoxicity with different degrees, the cell state is slightly worse than that of No. 1, No. 2 and No. 3 synthesized by the SINC group and that of No. 7, No. 8 and No. 9 synthesized by the invention, and the phenomena of cell debris, even cell cohesion and aging occur. From the test results in FIG. 6, the numbers 4 and 5 synthesized by the kit and the numbers 7, 8 and 9 synthesized by the invention all have obvious knock-down levels. No. 6 has no obvious target band and internal reference, and the analysis reason is that when the kit synthesizes siGAPDH and transfects 100pmol, the concentration is higher, the unknown impurities brought in by transfection are relatively increased, so that more cytotoxicity is caused, when the cells are collected for 48h, the cell number is reduced, the concentration of extracted protein is lower, and the No. 6 result is unstable. This also corresponds to the problem to be solved by the present invention. In comparison between No. 6 and No. 9, in the case of No. 9, siGAPDH synthesized according to the present invention was stable even when the transfection concentration was 100 pmol.

FIG. 7 is a grayscale histogram of Western Blot. The serial number writing completely corresponds to the sequence number in FIG. 6, namely No. 1, No. 2 and No. 3 are siNC, namely the comparison of non-interference efficiency sequences; no. 4, No. 5 and No. 6 are siGAPDH synthesized by the kit; no. 7, No. 8, No. 9 are siGAPD synthesized by the present invention; m is marker. Wherein the transfection concentrations No. 1, No. 4, No. 7 were 25 pmol; the transfection concentrations of Nos. 2, 5 and 8 were 50 pmol; the transfection concentrations of No. 3, No. 6 and No. 9 were 100 pmol. The analysis of the results is exactly the same as that of FIG. 6.

In summary, the products obtained by the current in vitro transcription methods or commercially available in vitro transcription kits contain too many impurities, which makes the experimental results very unstable. The method synthesizes siRNA through in vitro transcription of a DNA template with a T7 promoter; DNaseI degrades a DNA template, and S1 nuclease degrades single-stranded DNA and RNA; purifying and enriching siRNA by magnetic beads; the nucleic acid adsorption column further removes nucleic acid and base less than 20nt to obtain siRNA with high purity. Greatly improves the quality of siRNA preparation by in vitro transcription, improves the experimental efficiency and has very high popularization value.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.